Cytosolic forms of beta-lactamase and uses thereof

ABSTRACT

The present invention is directed to nucleic acid molecules that encode a cytosolic form of beta-lactamase and cells that include such nucleic acid molecules. These cells can be used in a variety of methods, such as methods for monitoring the expression of a gene.

This Application is a national stage application filed under 35 USC §371 of PCT/US96/04059, filed Mar. 20, 1996; which is acontinuation-in-part of U.S. application Ser. No. 08/407,544, filed Mar.20, 1995, which issued as U.S. Pat. No. 5,741,657, on Apr. 21, 1998.

This invention was made with Government support under Grant No. NS27177awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of chemistry andbiology. More particularly, the present invention relates tocompositions and methods for use in measuring gene expression.

A reporter gene assay measures the activity of a gene's promoter. Ittakes advantage of molecular biology techniques, which allow one to putheterologous genes under the control of any promoter and introduce theconstruct into the genome of a mammalian cell [Gorman, C. M. et al.,Mol. Cell Biol. 2: 1044-1051 (1982); Alam, J. and Cook, J. L., Anal.Biochem. 188: 245-254, (1990)]. Activation of the promoter induces thereporter gene as well as or instead of the endogenous gene. By designthe reporter gene codes for a protein that can easily be detected andmeasured. Commonly it is an enzyme that converts a commerciallyavailable substrate into a product. This conversion is convenientlyfollowed by either chromatography or direct optical measurement andallows for the quantification of the amount of enzyme produced.

Reporter genes are commercially available on a variety of plasmids forthe study of gene regulation in a large variety of organisms [Alam andCook, supra]. Promoters of interest can be inserted into multiplecloning sites provided for this purpose in front of the reporter gene onthe plasmid [Rosenthal, N., Methods Enzymol. 152: 704-720 (1987); Shiau,A. and Smith, J. M., Gene 67: 295-299 (1988)]. Standard techniques areused to introduce these genes into a cell type or whole organism [e.g.,as described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Expressionof cloned genes in cultured mammalian cells. In: Molecular Cloning,edited by Nolan, C. New York: Cold Spring Harbor Laboratory Press,1989]. Resistance markers provided on the plasmid can then be used toselect for successfully transfected cells.

Ease of use and the large signal amplification make this techniqueincreasingly popular in the study of gene regulation. Every step in thecascade DNA→RNA→Enzyme→Product→Signal amplifies the next one in thesequence. The further down in the cascade one measures, the more signalone obtains.

In an ideal reporter gene assay, the reporter gene under the control ofthe promoter of interest is transfected into cells, either transientlyor stably. Receptor activation leads to a change in enzyme levels viatranscriptional and translational events. The amount of enzyme presentcan be measured via its enzymatic action on a substrate. The substrateis a small uncharged molecule that, when added to the extracellularsolution, can penetrate the plasma membrane to encounter the enzyme. Acharged molecule can also be employed, but the charges need to be maskedby groups that will be cleaved by endogenous cellular enzymes (e.g.,esters cleaved by cytoplasmic esterases).

For a variety of reasons, the use of substrates which exhibit changes intheir fluorescence spectra upon interaction with an enzyme areparticularly desirable. In some assays, the fluorogenic substrate isconverted to a fluorescent product. Alternatively, the fluorescentsubstrate changes fluorescence properties upon conversion at thereporter enzyme. The product should be very fluorescent to obtainmaximal signal, and very polar, to stay trapped inside the cell.

To achieve the highest possible sensitivity in a reporter assay one hasto maximize the amount of signal generated by a single reporter enzyme.An optimal enzyme will convert 10⁵ substrate molecules per second undersaturating conditions [Stryer, L. Introduction to enzymes. In:Biochemistry, New York: W. H. Freeman and company, 1981, pp. 103-134].β-Lactamases will cleave about 10³ molecules of their favoritesubstrates per second [Chang, Y. H. et al., Proc. Natl. Acad. Sci. USA87: 2823-2827 (1990)]. Using a fluorogenic substrate one can obtain upto 10⁶ photons per fluorescent product produced, depending on the typeof dye used, when exciting with light of the appropriate wavelength. Thesignal terminates with the bleaching of the fluorophore [Tsien, R. Y.and Waggoner, A. S. Fluorophores for confocal microscopy: Photophysicsand photochemistry. In: Handbook of Biological Confocal Microscopy,edited by Pawley, J. B. Plenum Publishing Corporation, 1990, pp.169-178]. These numbers illustrate the theoretical magnitude of signalobtainable in this type of measurement. In practice a minute fraction ofthe photons generated will be detected, but this holds true forfluorescence, bioluminescence or chemiluminescence. A good fluorogenicsubstrate for a reporter enzyme has to have a high turnover at theenzyme in addition to good optical properties such as high extinctionand high fluorescence quantum yield.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide β-lactamasesubstrate compounds. It is a further object of the invention to providemembrane-permeant compounds. The membrane-permeant compounds may betransformed into substantially membrane-impermeant compounds.

Another object of the invention is to provide β-lactamase reportergenes. A further object of the present invention is to create cellscontaining the β-lactamase reporter genes functionally linked to apromotor such that when the promotor is turned on, the reporter genewill be expressed. Expression of the β-lactamase is measured with theβ-lactamase substrates which emit light after hydrolysis by theβ-lactamase.

A further object of the invention is to use the β-lactamase reportergenes in cells and the β-lactamase substrate compounds of the presentinvention to screen for biochemical activity.

In accordance with the present invention, fluorogenic substrates areprovided of the general formula I

wherein:

one of X and Y is a fluorescent donor moiety or a membrane-permeantderivative thereof, and the other is a quencher moiety, an acceptorfluorophore moiety or a membrane-permeant derivative thereof;

R′ is selected from the group consisting of H, lower alkyl, (CH₂)_(n)OH,(CH₂)_(n)COOR″, and ═NOJ, in which n is 0 or an integer from 1 to 5 andJ is H, Me, CH₂COOH, CHMeCOOH, and CMe₂COOH;

R″ is selected from the group consisting of H, physiologicallyacceptable metal and ammonium cations, —CHR²OCO(CH₂)_(n)CH₃,—CHR²OCOC(CH₃)₃, acylthiomethyl, acyloxy-alpha-benzyl,delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl,methylsulphinylmethyl, betamorpholinoethyl, dialkylaminoethyl,dialkylaminocarbonyloxymethyl, in which R² is selected from the groupconsisting of H and lower alkyl;

A is selected from the group consisting of S, O, SO, SO₂ and CH₂;

Z′ is a linker for X; and

Z″ is a linker for Y.

In another aspect, this invention provides methods for determiningwhether a sample contains β-lactamase activity. The methods involvecontacting the sample with a compound substrate of the invention, whichexhibits fluorescence resonance energy transfer when the compound isexcited; exciting the compound; and determining the degree offluorescence resonance energy transfer in the sample. A degree offluorescence resonance energy transfer that is lower than an expectedamount indicates the presence of β-lactamase activity. One embodiment ofthis method is for determining the amount of an enzyme in a sample.According to this method, determining the degree of fluorescenceresonance energy transfer in the sample comprises determining the degreeat a first and second time after contacting the sample with thesubstrate, and determining the difference in the degree of fluorescenceresonance energy transfer. The difference in the degree of fluorescenceresonance energy transfer reflects the amount of enzyme in the sample.

In another aspect, this invention provides recombinant nucleic acidmolecule comprising expression control sequences adapted for function ina vertebrate cell and operably linked to a nucleotide sequence codingfor the expression of a β-lactamase. It also provides recombinantnucleic acid molecules comprising expression control sequences adaptedfor function in a eukaryotic cell and operably linked to a nucleotidesequence coding for the expression of a cytosolic β-lactamase. Incertain embodiments, the invention is directed to mammalian host cellstransfected with these recombinant nucleic acid molecules.

In another aspect, this invention provides methods for determining theamount of β-lactamase activity in a cell. The methods involve providinga host cell transfected with a recombinant nucleic acid moleculecomprising expression control sequences operatively linked to nucleicacid sequences coding for the expression of a β-lactamase; contacting asample comprising the cell or an extract of the cell with a substratefor β-lactamase; and determining the amount of substrate cleaved,whereby the amount of substrate cleaved is related to the amount ofβ-lactamase activity.

In another aspect, this invention provides methods for monitoring theexpression of a gene operably linked to a set of expression controlsequences. The methods involve providing a host cell transfected with arecombinant nucleic acid molecule comprising expression controlsequences operatively linked to nucleic acid sequences coding for theexpression of a β-lactamase, except if the eukaryote is a fungus,wherein the β-lactamase is a cytosolic β-lactamase; contacting a samplecomprising the cell or an extract of the cell or conditioned medium witha substrate for β-lactamase; and determining the amount of substratecleaved. The amount of substrate cleaved is related to the amount ofβ-lactamase activity.

In another aspect, this invention provides methods for determiningwhether a test compound alters the expression of a gene operably linkedto a set of expression control sequences. The methods involve providinga cell transfected with a recombinant nucleic acid construct comprisingthe expression control sequences operably linked to nucleic acidsequences coding for the expression of a β-lactamase except if theeukaryote is a fungus, wherein the β-lactamase is a cytosolicβ-lactamase; contacting the cell with the test compound; contacting asample comprising the cell or an extract of the cell with a β-lactamasesubstrate; and determining the amount of substrate cleaved, whereby theamount of substrate cleaved is related to the amount of β-lactamaseactivity. In one embodiment of the methods, the substrate is a compoundof this invention. The step of determining the amount of substratecleaved comprises exciting the compound; and determining the degree offluorescence resonance energy transfer in the sample. A degree offluorescence resonance energy transfer that is lower than an expectedamount indicates the presence of β-lactamase activity.

In another aspect, this invention provides methods of clonal selectioncomprising providing cells transfected with a recombinant nucleic acidmolecule comprising the expression control sequences operably linked tonucleic acid sequences coding for the expression of a cytosolicβ-lactamase; contacting the cells with a substance that activates orinhibits the activation of the expression control sequences; contactingthe cells with a compound of claim 9 which is converted into asubstrate; determining whether substrate is cleaved within eachindividual cell, whereby cleavage reflects β-lactamase activity;selecting and propagating those cells with a selected level ofβ-lactamase activity. In a further embodiment, the method furtherinvolves culturing selected cells in the absence of activator for a timesufficient for cleaved substrate to be substantially lost from the cellsand for β-lactamase levels to return to unactivated levels; incubatingthe selected cells with a compound of claim 9 which is converted into asubstrate; and selecting cells that have not substantially cleaved thesubstrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) and 1(b) illustrate the emission spectra for the fluorescein(a) and rhodamine (b) components of compound 11 (Example 1) before andafter β-lactamase cleavage of the β-lactam ring;

FIG. 2 illustrates the emission spectrum of compound 17 before and afterβ-lactamase cleavage of the β-lactam ring;

FIG. 3 illustrates the emission spectrum of compound 22 before and afterβ-lactamase cleavage of the β-lactam ring;

FIG. 4 illustrates the emission spectrum of compound 25 before and afterβ-lactamase cleavage of the β-lactam ring;

FIG. 5 illustrates the emission spectrum of compound CCF2 before andafter β-lactamase cleavage of the β-lactam ring; and

FIG. 6 illustrates the emission spectrum of compound CCF1 before andafter β-lactamase cleavage of the β-lactam ring.

FIG. 7A presents a table describing various nucleotide and amino acidsequences useful in the invention.

FIGS. 7B-C depicts Sequence 1, the nucleotide and deduced amino acidsequence of E. coli RTEM as modified by Kadonaga et al (1984).

FIGS. 7D-E depicts Sequence 2, the nucleotide and deduced amino acidsequence of Wild-type secreted RTEM enzyme with Ser2→Arg, Ala23→Gly.

FIGS. 7F-G depicts Sequence 3, the nucleotide and deduced amino acidsequence of RTEM enzyme with β-globin upstream leader, mammalian Kozaksequence, replacement of signal sequence by Met Gly.

FIGS. 7H-I depicts Sequence 4, the nucleotide and deduced amino acidsequence of RTEM β-lactamase with mammalian Kozak sequence andreplacement of signal sequence by Met Asp.

FIGS. 7J-K depicts Sequence 5, the nucleotide and deduced amino acidsequence of Bacillus licheniformis β-lactamase with signal sequencereplaced.

DESCRIPTION OF THE INVENTION

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unless statedotherwise.

The term “fluorescent donor moiety” refers the radical of a fluorogeniccompound which can absorb energy and is capable of transferring theenergy to another fluorogenic molecule or part of a compound. Suitabledonor fluorogenic molecules include, but are not limited to, coumarinsand related dyes xanthene dyes such as fluoresceins, rhodols, andrhodamines, resorufins, cyanine dyes, bimanes, acridines, isoindoles,dansyl dyes, aminophthalic hydrazides such as luminol and isoluminolderivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans,aminoquinolines, dicyanohydroquinones, and europium and terbiumcomplexes and related compounds.

The term “quencher” refers to a chromophoric molecule or part of acompound which is capable of reducing the emission from a fluorescentdonor when attached to the donor. Quenching may occur by any of severalmechanisms including fluorescence resonance energy transfer,photoinduced electron transfer, paramagnetic enhancement of intersystemcrossing, Dexter exchange coupling, and exciton coupling such as theformation of dark complexes. The term “acceptor” as used herein refersto a quencher which operates via fluorescence resonance energy transfer.Many acceptors can re-emit the transferred energy as fluorescence.Examples include coumarins and related fluorophores, xanthenes such asfluoresceins, rhodols, and rhodamines, resorufins, cyanines,difluoroboradiazaindacenes, and phthalocyanines. Other chemical classesof acceptors generally do not re-emit the transferred energy. Examplesinclude indigos, benzoquinones, anthraquinones, azo compounds, nitrocompounds, indoanilines, di- and triphenylmethanes.

The term “dye” refers to a molecule or part of a compound which absorbsspecific frequencies of light, including but not limited to ultravioletlight. The terms “dye” and “chromophore” are synonymous.

The term “fluorophore” refers to chromophore which fluoresces.

The term “membrane-permeant derivative” means a chemical derivative of acompound of general formula wherein at least one of X and Y contains atleast one acylated aromatic hydroxyl, acylated amine, or alkylatedaromatic hydroxyl wherein the acyl group contains 1 to 5 carbon atomsand wherein the alkyl group is selected from the group consisting of—CH₂OC(O)alk, —CH₂SC(O)alk, —CH₂OC(O)Oalk, lower acyloxy-alpha-benzyl,and deltabutyrolactonyl; wherein alk is lower alkyl of 1 to 4 carbonatoms. These derivatives are made better able to cross cell membranes,i.e. membrane permeant, because hydrophilic groups are masked to providemore hydrophobic derivatives. Also, the masking groups are designed tobe cleaved from the fluorogenic substrate within the cell to generatethe derived substrate intracellularly. Because the substrate is morehydrophilic than the membrane permeant derivative it is now trappedwithin the cells.

The term “alkyl” refers to straight, branched, and cyclic aliphaticgroups of 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, and mostpreferably 1 to 4 carbon atoms. The term “lower alkyl” refers tostraight and branched chain alkyl groups of 1 to 4 carbon atoms.

The term “aliphatic” refers to saturated and unsaturated alkyl groups of1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and mostpreferably 1 to 4 carbon atoms.

SUBSTRATES

β-Lactamases are nearly optimal enzymes in respect to their almostdiffusion-controlled catalysis of β-lactam hydrolysis [Christensen, H.et al., Biochem. J. 266: 853-861 (1990)]. Upon examination of the otherproperties of this class of enzymes, it was determined that they weresuited to the task of an intracellular reporter enzyme. They cleave theβ-lactam ring of β-lactam antibiotics, such as penicillins andcephalosporins, generating new charged moieties in the process[O'Callaghan, C. H. et al., Antimicrob. Agents. Chemother. 8: 57-63,(1968); Stratton, C. W., J. Antimicrob. Chemother. 22, Suppl. A: 23-35(1988)]. A first generation cephalosporin is illustrated below, left,with the arrow pointing to the site of cleavage by β-lactamase. The freeamino group thus generated (middle structure below) donates electrondensity through the vinyl group to promote irreversible cleavage of anucleofugal group R₂ from the 3′-position. R₂ is thus free to diffuseaway from the R₁-cephalosporin conjugate (right-hand structure below).

β-Lactamases are a class of enzymes that have been very wellcharacterized because of their clinical relevance in making bacteriaresistant to β-lactam antibiotics [Waley, S. G., Sci. Prog. 72: 579-597(1988); Richmond, M. H. et al., Ann. N.Y. Acad. Sci. 182: 243-257(1971)]. Most β-lactamases have been cloned and their amino acidsequence determined [see, e.g., Ambler, R. P., Phil. Trans. R. Soc.Lond. [Ser.B.] 289: 321-331 (1980)].

A gene encoding β-lactamase is known to molecular biologists as theampicillin resistance gene (Amp^(r)) and is commonly used to select forsuccessfully transduced bacteria [Castagnoli, L. et al., Genet. Res. 40:217-231 (1982)]; clones thereof are almost universally available. Theenzyme catalyzes the hydrolysis of a β-lactam ring and will not acceptpeptides or protein substrates [Pratt, R. F. and Govardhan, C. P., Proc.Natl. Acad. Sci. USA 81: 1302-1306 (1984); Murphy, B. P. and Pratt, R.F., Biochemistry 30: 3640-3649 (1991)]. The kinetics of this reaction iswell understood and there is no product inhibition [Bush, K. and Sykes,R. B., Antimicrob. Agents. Chemother. 30: 6-10 (1986); Christensen etal. (1990), supra]. The enzyme substrates are less polar than theproducts.

The carboxyl group in the substrate can be easily masked by anacetoxymethyl ester [Jansen, A. B. A. and Russell, T. J., J. Chem. Soc.2127-2132, (1965); Daehne, W. et al., J. Med. Chem. 13: 607-612 (1970)],which is readily cleaved by endogenous mammalian intracellularesterases. Conversion by these esterases followed by the β-lactamcleavage by β-lactamase generates two negative charges and a tertiaryamine, which protonates. To date, there has been no report of afluorogenic substrate with the appropriate properties, but multiplechromogenic substrates of different design have been reported and arecommercially available [Jones, R. N. et al., J. Clin. Microbiol. 15:677-683 (1982); Jones, R. N. et al., J. Clin. Microbiol. 15: 954-958(1982); O'Callaghan, C. H. et al., Antimicrob. Agents. Chemother. 1:283-288 (1972)].

A large number of β-lactamases have been isolated and characterized, allof which would be suitable for use in accordance with the presentinvention. Initially, β-lactamases were divided into different classes(I through V) on the basis of their substrate and inhibitor profiles andtheir molecular weight [Richmond, M. H. and Sykes, R. B., Adv. Microb.Physiol. 9: 31-88 (1973)]. More recently, a classification system basedon amino acid and nucleotide sequence has been introduced [Ambler, R.P., Phil. Trans. R. Soc. Lond. [Ser.B.] 289: 321-331 (1980)]. Class Aβ-lactamases possess a serine in the active site and have an approximateweight of 29 kd. This class contains the plasmid-mediated TEMβ-lactamases such as the RTEM enzyme of pBR322. Class B β-lactamaseshave an active-site zinc bound to a cysteine residue. Class C enzymeshave an active site serine and a molecular weight of approximately 39kd, but have no amino acid homology to the class A enzymes.

The coding region of an exemplary β-lactamase employed in the reportergene assays described herein is indicated in SEQ ID NO:1 (nucleic acidsequence) and SEQ ID NO:2 (amino acid sequence). The pTG2del1 containingthis sequence has been described [Kadonaga, J. T. et al., J. Biol. Chem.259: 2149-2154 (1984)]. The entire coding sequence of wildtype pBR322β-lactamase has also been published [Sutcliffe, J. G., Proc. Natl. Acad.Sci. USA 75: 3737-3741 (1978)]. As would be readily apparent to thoseskilled in the field, this and other comparable sequences for peptideshaving β-lactamase activity would be equally suitable for use inaccordance with the present invention. The β-lactamase reporter gene isemployed in an assay system in a manner well known per se for the use ofreporter genes (for example, in the form of a suitable plasmid vector).

In conjunction with a suitable β-lactamase, there are employed inaccordance with the present invention fluorogenic substrates of thegeneral formula I

in which one of X and Y is a fluorescent donor moiety and the other is aquencher (which may or may not re-emit); R′ is selected from the groupconsisting of H, lower alkyl, (CH₂)_(n)OH, (CH₂)_(n)COOR″, and ═NOJ, inwhich n is 0 or an integer from 1 to 5 and J is H, Me, CH₂COOH,CHMeCOOH, and CMe₂COOH; R″ is selected from the group consisting of H,physiologically acceptable metal and ammonium cations,—CHR²OCO(CH₂)_(n)CH₃, —CHR²OCOC(CH₃)₃, acylthiomethyl,acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl,phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl,and dialkylaminocarbonyloxymethyl, in which R² is selected from thegroup consisting of H and lower alkyl; A is selected from the groupconsisting of S, O, SO, SO₂ and CH₂; and Z′ and Z″ are linkers for thefluorescent donor and quencher moieties.

The linkers Z′ and Z″ serve the purpose of attaching the fluorescentdonor and quencher moieties to the cephalosporin-derived backbone, andmay facilitate the synthesis of the compounds of general formula I. Ingeneral formula I, Z′ may represent a direct bond to the backbone;alternatively, suitable linkers for use as Z′ include, but are notlimited to, the following: —(CH₂)_(n)CONR²(CH₂)_(m)—,—(CH₂)_(n)NR²CO(CH₂)_(m)—, —(CH₂)_(n)NR³CONR²(CH₂)_(m)—,—(CH₂)_(n)NR³CSNR²(CH₂)_(m)—, —(CH₂)_(n)CONR³(CH₂)_(p)CONR²(CH₂)_(m)—,—(CH₂)_(n)—, —(CH₂)_(n)NR³CO(CH₂)_(p)S(CH₂)_(m)—, —(CH₂)_(n)S(CH₂)_(m)—,—(CH₂)_(n)O(CH₂)_(m)—, —(CH₂)_(n)NR²(CH₂)_(m)—,—(CH₂)_(n)SO₂NR²(CH₂)_(m)—, —(CH₂)_(n)CO₂(CH₂)_(m)—,

wherein R² and n are as previously defined; R³ is selected from thegroup consisting of hydrogen and lower alkyl; and each of m and p isindependently selected from the group consisting of 0 and integers from1 to 4. Especially preferred are Z′ groups such where n and m are 0.Also particularly preferred are such Z′ groups where R² is H.

Suitable linkers Z″ for the Y moiety include, but are not limited to, adirect bond to a heteroatom (e.g., O, N or S) in the dye's chromophoreor the following: —O(CH₂)_(n)—, —S(CH₂)_(n)—, —NR²(CH₂)_(n)—, —N⁺R²₂(CH₂)_(n)—, —OCONR²(CH₂)_(n)—, —O₂C(CH₂)_(n)—, —SCSNR²(CH₂)_(n)—,—SCSO(CH₂)_(n)—, —S(CH₂)_(n)CONR²(CH₂)_(m), —S(CH₂)_(n)NR²CO(CH₂)_(m),and

in which R², n and m are as previously defined; and m is an integer from0 to 4. Particularly preferred Z″ groups are —S(CH₂)_(n). Especiallypreferred is H.

Preferred R′ groups include H and methyl. Particularly preferred is H.Preferred R″ groups include H and acetoxymethyl. A preferred R² group isH. A preferred A group is —S—.

In a preferred aspect, the compounds of the present invention aremembrane-permeant. Particularly preferred are such compounds wherein atleast one of X and Y contains at least one acylated aromatic hydroxyl,acylated amine, or alkylated aromatic hydroxyl wherein the acyl groupcontains 1 to 5 carbon atoms and wherein the alkyl group is selectedfrom the group consisting of —CH₂OC(O)alk, —CH₂SC(O)alk, —CH₂OC(O)Oalk,lower acyloxy-alpha-benzyl, and delta-butyrolactonyl, wherein alk islower alkyl of 1 to 4 carbon atoms. Particularly preferred are suchcompounds where at least one of X and Y contains at least one acylatedaromatic hydroxy, wherein the acyl group is either acetyl, n-propionyl,or n-butyryl. Also particularly preferred are such compounds wherein atleast one of X and Y contains an acetoxy methyl group on an aromatichydroxyl group.

In another preferred aspect, the quencher or acceptor is a fluorescein,rhodol, or rhodamin of formulae VIII-XII. Preferred are such compoundswhere the donor is a fluorescein of formula VIII and the quencher oracceptor is a rhodol or rhodamine of formulae VIII-XII. Also preferredare such compounds where the donor is a fluorescein of formula VIII andthe quencher or acceptor is a tetrahalo fluorescein of formula VIII inwhich R^(a), R^(b), R^(c), and R^(d) are independently Br or Cl. Alsopreferred are such compounds where the quencher or acceptor is a rhodolof formulae VIII, IX, and XI. Another preferred group of such compoundsare those where the quencher or acceptor is a rhodamine of formulaeVIII, X, and XII.

In a another preferred aspect, the donor is a coumarin of formulaeII-VII and the quencher/acceptor is a fluorescein, rhodol, or rhodamineof formulae VIII-XII, XLVII, or XLVII, and membrane-permeant fluorogenicderivatives thereof. Particularly preferred are such compounds with afluorescein quencher/acceptor of formula VIII. Especially preferred aresuch compounds where the coumarin is 7-hydroxycoumarin or7-hydroxy-6-chlorocoumarin and the fluorescein acceptor is fluoresceinor dichlorofluorescein.

As would readily be appreciated by those skilled in the art, theefficiency of fluorescence resonance energy transfer depends on thefluorescence quantum yield of the donor fluorophore, the donor-acceptordistance and the overlap integral of donor fluorescence emission andacceptor absorption. The energy transfer is most efficient when a donorfluorophore with high fluorescence quantum yield (preferably, oneapproaching 100%) is paired with an acceptor with a large extinctioncoefficient at wavelengths coinciding with the emission of the donor.The dependence of fluorescence energy transfer on the above parametershas been reported [Forster, T. (1948) Ann. Physik 2: 55-75; Lakowicz, J.R., Principles of Fluorescence Spectroscopy, New York: Plenum Press(1983); Herman, B., Resonance energy transfer microscopy, in:Fluorescence Microscopy of Living Cells in Culture, Part B, Methods inCell Biology, Vol 30, ed. Taylor, D. L. & Wang, Y. -L., San Diego:Academic Press (1989), pp. 219-243; Turro, N. J., Modern MolecularPhotochemistry, Menlo Part: Benjamin/Cummings Publishing Co., Inc.(1978), pp. 296-361], and tables of spectral overlap integrals arereadily available to those working in the field [for example, Berlman,I. B. Energy transfer parameters of aromatic compounds, Academic Press,New York and London (1973)]. The distance between donor fluorophore andacceptor dye at which fluorescence resonance energy transfer (FRET)occurs with 50% efficiency is termed R₀ and can be calculated from thespectral overlap integrals. For the donor-acceptor pairfluorescein-tetramethyl rhodamine which is frequently used for distancemeasurement in proteins, this distance R₀ is around 50-70 Å [dosRemedios, C. G. et al. (1987) J. Muscle Research and Cell Motility8:97-117]. The distance at which the energy transfer in this pairexceeds 90% is about 45 Å. When attached to the cephalosporin backbonethe distances between donors and acceptors are in the range of 10 Å to20 Å, depending on the linkers used and the size of the chromophores.For a distance of 20 Å, a chromophore pair will have to have acalculated R₀ of larger than 30 Å for 90% of the donors to transfertheir energy to the acceptor, resulting in better than 90% quenching ofthe donor fluorescence. Cleavage of such a cephalosporin by β-lactamaserelieves quenching and produces an increase in donor fluorescenceefficiency in excess of tenfold. Accordingly, it is apparent thatidentification of appropriate donor-acceptor pairs for use as taughtherein in accordance with the present invention would be essentiallyroutine to one skilled in the art.

To measure β-lactamase activity in the cytoplasm of living cells,smaller molecular weight chromophores as hereinafter described are ingeneral preferred over larger ones as substrate delivery becomes aproblem for larger compounds. Large molecules, especially those overabout 1200 daltons, also tend to bind more avidly to cellularconstituents than small ones, thereby removing at least some of themfrom access and cleavage by β-lactamase.

Chromophores suitable for use as X and Y are well known to those skilledin the art. Generic structures of particular classes of chromophoressuitable for use as X and Y are provided below. Compounds of generalformulas II-XXXIV are exemplary of fluorophores which serve as the basisfor particularly suitable donor moieties in the compounds of generalformula I. Suitable chromophores for use as the basis of acceptormoieties in the compounds of general formula include, but are notlimited to, compounds of general formulas II-LIV. Chromophores ofgeneral formulae XXXV-LIV usually do not re-emit efficiently.

In preferred embodiments of the compounds of general formulas II-LVI:

each of a and a′ is independently H or an attachment point (i.e., alocation at which the dye moiety is attached to the core structure ofgeneral formula I;

E is selected from the group consisting of H, OH, OR^(k) andNR^(g)R^(h);

G is selected from the group consisting of O and N⁺R^(g′)R^(h′);

each of L and L′ is independently selected from the group consisting ofCH and N;

M is selected from the group consisting of H, Mg, Al, Si, Zn, and Cu;

Q is selected from the group consisting of O, S, C(CH₃)₂ and NR^(g);

Q′ is selected from the group consisting of O, CH₂, C(CH₃)₂, NR^(k) andSO₂;

T is selected from the group consisting of O and NR^(k);

each of W and W′ is selected from the group consisting of O, S, Se andNH;

each of R^(a), R^(b), R^(c) and R^(d) is independently selected from thegroup consisting of an attachment point, H, halogen and lower alkyl;

R^(e) is selected from the group consisting of an attachment point, H,lower alkyl, (CH₂)_(n)CO₂H, (CH₂)_(n)CHaCO₂H, CHa(CH₂)_(n)CO₂H,(CH₂)_(n)COa, CH=CHCOa,

each of R^(f), R^(g), R^(g′), R^(h), R^(h) and R^(k) is independentlyselected from the group consisting of an attachment point, H, loweralkyl and CH₂(CH₂)_(n)a;

R^(i) is selected from the group consisting of an attachment point, H,halogen, lower alkyl, CN, CF₃, phenyl, CO₂H and CONR^(g′)R^(h′);

R^(j) is selected from the group consisting of an attachment point, H,halogen, lower alkyl, CN, CF₃, phenyl, CH₂CO₂H, CH₂CONR^(g′)R^(h′);

each of R¹ and R^(r) is independently selected from the group consistingof an attachment point, H, lower alkyl,

each of R^(m), R^(n), R^(p) and R^(q) is independently selected from thegroup consisting of an attachment point, H, lower alkyl and phenyl;

R^(o) is selected from the group consisting of an attachment point, Hand lower alkyl;

each of R^(s) and R^(t) is independently selected form the groupconsisting of an attachment point, H, halogen, lower alkyl and OR^(f);

each of R^(u) and R^(v) is independently selected from the groupconsisting of an attachment point, H, halogen, CN and NO₂;

each of R^(w) is independently selected from the group consisting of anattachment point, H, COO⁻, SO₃ ⁻, and PO₃ ²⁻

Ln is selected from the group consisting of Eu³⁺, Ln³⁺, and Sm³⁺,

Chel is a polydentate chelator with at least six and preferably eight toten donor atoms that can face into a cavity of diameter between 4 and 6angstroms, which may or may not be macrocyclic, which includes achromophore absorbing between 300 and 400 nm, and which includes anattachment point through which Chel can be conjugated to Z′ or Z″. Asuitable Chel moiety is a europium tris-(bipyridine) cryptands. In theanthraquinone chromophores of general formula XXXIX, each of positions1-8 may carry a substituent H or E, or serve as an attachment point.

Europium tris-(bipyridine) cryptand donors may be suitably paired withacceptors of the formulae XV-XVII, XXXVI, XLVI-XLVII, LIV and LVI.Terbium tris-(bipyridine) cryptand donors may be suitably paired withacceptors of the formulae VIII-XVIII, XXXVI-XLI, and XLV-LIV, and VLI.

The Europium tris-(bipyridine) cryptand/phtalocyanines donor/acceptorpair may be of particular interest when it is desirable to measureβ-lactamase activity by emission of energy in the near to far red range.

In many applications it is desirable to derivatize compounds of generalformula I to render them hydrophobic and permeable through cellmembranes. The derivatizing groups should undergo hydrolysis insidecells to regenerate the compounds of general formula I and trap theninside the cells. For this purpose, it is preferred that any phenolichydroxyls or free amines in the dye structures are acylated with C₁-C₄acyl groups (e.g. formyl, acetyl, n-butryl) or converted to variousother esters and carbonates [for examples, as described in Bundgaard,H., Design of Prodrugs, Elsevier Science Publishers (1985), Chapter I,page 3 et seq.]. Phenols can also be alkylated with 1-(acyloxy)alkyl,acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, ormethoxycarbonyloxymethyl groups. In the case of fluoresceins, rhodols,and rhodamines this manipulation is particularly useful, as it alsoresults in conversion of the acid moiety in these dyes to thespirolactone. To promote membrane permeation, the carboxyl at the4-position of the cephalosporin should be esterified with1-(acyloxy)alkyl, acylthiomethyl, acyloxy-alpha-benzyl,delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl,methylsulfinylmethyl, beta-morpholionethyl, 2-(dimethylamino)ethyl,2-(diethylamino)ethyl, or dialkylaminocarbonyloxymethyl groups asdiscussed in Ferres, H. (1980) Chem. Ind. 1980:435-440. The mostpreferred esterifying group for the carboxyl is acetoxymethyl.

A general method for synthesis of compounds of general formula I isdepicted below. As one of ordinary skill in the art will appreciate, themethods below can be used for a variety of derivatives, and othermethods of synthesis are possible.

In these compounds, RG is a nucleophile-reactive group (e.g.,iodoacetamide, isocyanate, isothiocyanate, etc.); Nu is a nucleophile(e.g., —SH, —NH₂, —OH, etc.); R₀ is H or an ester group (e.g.,benzhydryl ester, tertiary butyl ester, etc.); Nu₀ is a bidentatenucleophile (e.g., HS⁻, HSCH₂CH₂NH₂, xanthate, etc.); and Hal is ahalogen (e.g., chlorine, bromine or iodine).

The cephalosporin starting materials are commercially availablecephalosporin derivatives 7-aminocephalosporanic acid or 7-amino3′-chloro-cephalosporanic acid as its benzhydryl or tertiary butyl ester(R₀). Prior to coupling the dyes A and B carrying nucleophile reactivegroups (RG) it is sometimes advantageous to esterify or alkylate theirphenolic and free amine residues. The order of attaching dye A and dye Bdepends on the choice of reagents. Dye A is tethered to thecephalosporin via an alkyl amide linker. This is achieved by reacting adye A carrying a nucleophile-reactive group (RG) with a bifunctionalaliphatic acid (e.g., amino-, mercapto- or hydroxyalkyl acid) andcoupling of the acid to the cephalosporin 7-amine (path 1).Alternatively, dye A carrying a nucleophilic group (e.g., amine orthiol) is reacted with a halogenated alkyl acid and the acid coupled tothe cephalosporin 7-amine (path 2). In both pathways, the order of thetwo reactions can be reversed. Dyes A containing an aliphatic acid canbe directly coupled to the cephalosporin (path 3). Dye B carrying anucleophilic substituent can be coupled to the 3′-position in thecephalosporin by direct displacement of the leaving group (LG) (path 4).A Dye B carrying a nucleophile-reactive group can be reacted with abidentate nucleophile which is coupled then attached to thecephalosporin by leaving group (LG) displacement (path 5); the order ofthe reactions can be reversed.

In some cases it might be necessary to conduct the first reaction with abidentate nucleophile with one of its nucleophilic groups masked. Thesecond coupling is then performed after removal of that protectiongroup. After attachment of both dyes the cephalosporin ester is cleaved(in cases where R₀ is not H). To make membrane permeant substrates theacid is then re-esterified to esters that can be deprotected by thecytoplasmic environment of a mammalian cell. For applications notinvolving cell cytoplasm, any remaining acyl and alkyl groups that wereused to mask phenols and free amines on the dyes are removed.

Preferred combinations of classes of donors and acceptors suitable foruse in accordance with the present invention are indicated in Table 1.In embodiments of compounds of general formula I using thesecombinations, fluorescent resonant energy transfer (FRET) occurs. Ofcourse, as would be readily understood by those working in the field,many other combinations of donors and acceptors/quenchers (includingthose that re-emit and those that do not) would be suitable for use inaccordance with the present invention. In general, suitable donor andacceptor pairs are those where the donor's emission spectrumsignificantly overlaps the acceptor's excitation spectrum.

TABLE 1 DONORS II-VIII, XIX-XXI, VII-XIV, ACCEPTORS XXIII-XXXIV XVII,XXII XV-XVI, LV II-VIII, FRET XIX-XXI, XXIII- XXXIV VII-XIV, FRET FRETXVII, XXII XV-XVII FRET FRET FRET XL-XLV, FRET FRET XLVII-LIIXXXV-XXXIX, FRET FRET FRET XLVI-XLVII, LIII-LIV, LVI

Fluorescent donor moieties of particular interest include coumarins andfluoresceins. Particular quenchers of interest include fluoresceins,rhodols and rhodamines. Combinations of interest include the use of acoumarin donor with a fluorescein, rhodol or rhodamine quencher, and afluorescein donor with a rhodol or rhodamine quencher. Specificcombinations of interest include the following: a coumarin (e.g.,7-hydroxycoumarin) or chloro derivative thereof with a fluorescein ordichloro derivative thereof; a fluorescein with an eosin ortetrachlorofluorescein; a fluorescein with a rhodol derivative; and arhodamine with a fluorescein.

Europium chelate donors may be suitably paired with acceptors of theformulae XV-XVII, XXXVI, XLVI-XLVII, LIV, and LVI. Terbium chelatedonors may be suitably paired with acceptors of the formulae VIII-XVIII,XXXVI-XLI, and XLV-LIV, and LVI. The europium and terbium chelate donorsmay be of particular interest for their very narrow emission peaks andtheir microsecond-to-millisecond excited state lifetimes, which can bereadily discriminated from background fluorescence and scattering withexcited-state lifetimes of nanoseconds or less.

In many applications it is desirable to derivatize compounds of generalformula I to render them more hydrophobic and permeable through cellmembranes. The derivatizing groups should undergo hydrolysis insidecells to regenerate the compounds of general formula I and trap theminside the cells. For this purpose, it is preferred that any phenolichydroxyls or free amines in the dye structures are acylated with C₁-C₄acyl groups (e.g. formyl, acetyl, n-butyryl) or converted to variousother esters and carbonates [for example, as described in Bundgaard, H.,Design of Prodrugs, Elsevier Science Publishers (1985), Chapter 1, page3 et seq.]. Phenols can also be alkylated with 1-(acyloxy)alkyl,acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, ormethoxycarbonyloxymethyl groups. In the case of fluoresceins, rhodolsand rhodamines, acylation or alkylation of the free phenolic groups isparticularly useful, as it also results in conversion of the acid moietyin these dyes to the spirolactone. To promote membrane permeation, thecarboxyl at the 4-position of the cephalosporin should be esterifiedwith 1-(acyloxy)alkyl, acylthiomethyl, acyloxy-alpha-benzyl,delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl,methylsulfinylmethyl, beta-morpholinoethyl, 2-(dimethylamino)ethyl,2-(diethylamino)ethyl, or dialkylaminocarbonyloxymethyl groups asdiscussed in Ferres, H. (1980) Chem. Ind. 1980:435-440. The mostpreferred esterifying group for the carboxyl is acetoxymethyl.

The cephalosporin backbone serves as a cleavable linker between twodyes. After cleavage it provides the charges necessary to keep one ofthe two dyes inside the cell. Dyes are chosen in a manner that one dyeabsorbs light (quencher or acceptor chromophore) at the wavelength thatthe other one emits (donor fluorophore). In the intact cephalosporin thetwo dyes are in close proximity to each other. When exciting the donorfluorophore one observes fluorescence resonance energy transfer (FRET)from the donor to the acceptor instead of donor fluorescence [Forster,T., Ann. Physik 2:55-75 (1948)]. If the acceptor is a nonfluorescent dyethe energy is given off to the solvent; the donor fluorescence isquenched. In the case of the acceptor being itself a fluorescent dye,fluorescence re-emission occurs at the acceptor's emission wavelength.In polar solvents such as water, hydrophobic donor and acceptorfluorophores can stack when separated by a short flexible linker. Due tothis association in the ground state, a “dark complex” is formed [Yaron,A. et al., Anal. Biochem. 95:228-235 (1979)]. In this complex, neitherfluorophore can emit light, causing the fluorescence of both dyes to bequenched [Bojarski, C. and Sienicki, K. Energy transfer and migration influorescent solutions. In: Photochemistry and Photophysics, edited byRabek, J. F. Boca Raton: CRC Press, Inc., 1990, pp. 1-57]. In eithercase, a large change in fluorescence goes along with β-lactam cleavage,which can be used to measure β-lactamase activity. As both dyes diffuseaway from each other, stacking and energy transfer are disrupted.Cephalosporins carrying a donor and an acceptor dye which fluoresces arereferred to herein as FRET-cephalosporins.

Fluorescence resonance energy transfer has been used as a spectroscopicruler for measuring molecular distances in proteins and peptides as itis effective in the range from 10-100 Å. This energy transfer isproportional to the inverse sixth power of the distance between donorand acceptor. Its efficiency is higher, the better donor emission andacceptor absorbance overlap, and the longer the fluorescence lifetime ofthe donor (in absence of the acceptor). FRET can be very efficient overdistances of 10-20 Å.

In the cephalosporin, distances for attachment of donor and acceptor aregreater than 10 Å and a minimum of 10 bond-lengths, if one includes thetwo minimal spacers at 7- and 3-positions. Over this distance FRET isvery efficient, if the right donor-acceptor pairs are chosen.Conveniently, in a FRET-cephalosporin the 7-amine tethered dye staysattached to the polar hydrolysis products of cephalosporin cleavage,trapping it in the cells' cytoplasm. This position is best occupied bythe donor fluorophore, although in some instances the acceptor mayoccupy this position. Upon cleavage, fluorescence increases due to lossof the quencher dye.

The acceptor fluorophore is generally attached by a linker which impartsthe greatest stability of the substrate to nucleophilic attack. Apreferred linker is a thioether bond (—S—), which is very stable and dueto its inductive effect reduces the reactivity of the β-lactam ringtoward nucleophiles [Page, M. I. Adv. Phys. Org. Chem. 23:165-270(1987)]. In addition, the free thiol or thiolate group released uponhydrolysis often quenches the attached fluorophore, adding to thedesired large change in fluorescence upon hydrolysis.

The fluorogenic substrates of the invention are initially colorless andnonfluorescent outside cells. The substrates are designed so theyreadily cross cell membranes into the cytoplasm, where they areconverted to fluorescent compounds by endogenous nonspecific esterasesand stay trapped due to their charges. In the intact molecules,fluorescence energy transfer occurs leading to fluorescence at aparticular wavelength when the substrates are excited. Lactamasecleavage of the β-lactam ring is followed by expulsion of thefluorescein moiety with loss of fluorescence now results in fluorescenceat a different wavelength.

The assay systems of the present invention further provide anadvantageous and rapid method of isolation and clonal selection ofstably transfected cell lines containing reporter genes and having thedesired properties which the transfection was intended to confer, e.g.fluorescent signal response after activation of a transfected receptorwith a high signal-to-noise ratio from a high proportion of isolatedcells. Current procedures for clonal selection of satisfactorilytransfected, genetically engineered cells from the initial population,are done mainly by replica plating of colonies, testing of one set ofcolonies, visual selection of preferred clones, manual isolation of thereplicas of the preferred clones by pipetting, and prolonged cellularcultivations. This procedure is laborious and time-consuming; it mayrequire several months to generate a clone useful for assays suited todrug screening. Moreover, it is difficult to manually select andmaintain more than a few hundred clones. Using the assays of thispresent invention, the desired signal from cellular beta-lactamasereporter system can be maintained within living and viable cells.Replica plating of colonies is unnecessary because single cells can beassayed and remain viable for further multiplication. Thus, from thepopulation of initially transfected cells, one can rapidly select thosefew inidividual living cells with the best fluorescent signal, usingautomated instruments such as a fluorescent-activated cell sorter, e.g.the Becton Dickinson FACS Vantage™. The selected cells are thencollected for cultivation and propagation to produce a clonal cell linewith the desired properties for assays and drug screening.

As would be immediately apparent to those working in the field, thecombination of a novel substrate in accordance with the invention and asuitable β-lactamase may be employed in a wide variety of differentassay systems (such as are described in U.S. Pat. No. 4,740,459). Inparticular the fluorogenic substrates of the invention enable thedetection of β-lactamase activity in a wide variety of biologicallyimportant environments, such as human blood serum, the cytoplasm ofcells and intracellular compartments; this facilitates the measurementof periplasmic or secreted β-lactamase.

Further, the expression of any target protein can be detected by fusinga gene encoding the target protein to a β-lactamase gene, which can belocalized by immunostaining and fluorescence or electron microscopy. Forexample, β-lactamase fusion proteins may be detected in the lumen oforganelles through the use of the substrates of the invention; onlysubcellular compartments containing the fusion protein fluoresce at awavelength characteristic of the cleaved substrate, whereas all othersfluoresce at a wavelength characteristic of the intact molecule.

Both the intact and cleaved substrate are well retained in cells withoutthe use of special measures, such as chilling. The color change (even inindividual small mammalian cells) is visible through a fluorescencemicroscope using normal color vision or photographic film; thefluorescence signal may be quantified and further enhanced byconventional digital image processing techniques. Moreover, because geneactivation is detected not by a change in a single intensity but ratherby a color change or a change in the ratio between two intensities atdifferent wavelengths, the assays of the present invention arerelatively immune to many artifacts such as variable leakiness of cells,quantity of substrate, illumination intensity, absolute sensitivity ofdetection and bleaching of the dyes.

A variety of substrates (e.g., compounds of general formulas 17, 22 and25) have been prepared and their emission spectra obtained before andafter β-lactamase cleavage. These substrates allow for β-lactamasedetection primarily in vitro, as they bind strongly to serum andcellular proteins. Due to their hydrophobic nature, the fluorophoresstack; this leads to a loss of fluorescence in the intact substrate.β-lactamase cleaves the substrates and relieves the stacking, allowingfor fluorescence. Compounds (e.g., compound 11, Example 1) with reversedlocation of donor and acceptor fluorophore on the cephalosporin exhibitsimilar fluorescence behavior.

In one preferred embodiment of the invention, a compound of generalformula 1 was coupled to a compound of general formula 2 to form acompound of general formula 3. Commercially-available compound 4 wasthen coupled to compound 3 using dicyclohexylcarbodiimide and theproduct reacted with compound 5, yielding a compound of general formula6. Deprotection of compound 6 generated a compound of general formula 7.In exemplary embodiments, Acyl was acetyl, R^(x) was Me and R^(y) H (a),or Acyl was butyryl, R^(x) was H and R^(y) Cl (b); R^(z) wastrimethylsilyl or benzyl.

The compounds of general formula 6 were modified to obtain membranepermeant derivatives which were converted to the correspondingfluorescent compounds of general formula 7 in intact cells due to theaction of endogenous nonspecific esterases. In these molecules,fluorescence resonance energy transfer occurs from the 7-hydroxycoumarinmoiety to the fluorescein moiety, leading to green fluorescence when thecompounds are excited at about 400 nm. After cleavage of the β-lactamring, excitation of the 7-hydroxycoumarin moiety results in bluefluorescence; in exemplary embodiments, a 25-fold increase influorescence at about 450 nm and a three- to fourfold decrease influorescence at 515 nm was observed.

MONITORING GENE EXPRESSION

The substrates of this invention make it feasible to use β-lactamase asa reporter gene to monitor the expression from a set of expressioncontrol sequences. In one aspect, this invention provides methods formonitoring gene expression from a set of expression control sequences byusing β-lactamase as a reporter gene. A cell is provided that has beentransfected with a recombinant nucleic acid molecule comprising theexpression control sequences operably linked to nucleic acid sequencescoding for the expression of β-lactamase.

Recombinant Nucleic Acids

As used herein, the term “nucleic acid molecule” includes both DNA andRNA molecules. It will be understood that when a nucleic acid moleculeis said to have a DNA sequence, this also includes RNA molecules havingthe corresponding RNA sequence in which “U” replaces “T”. The term“recombinant nucleic acid molecule” refers to a nucleic acid moleculewhich is not naturally occurring, and which comprises two nucleotidesequences which are not naturally joined together. Recombinant nucleicacid molecules are produced by artificial combination, e.g., geneticengineering techniques or chemical synthesis.

Nucleic acids encoding β-lactamases can be obtained by methods known inthe art, for example, by polymerase chain reaction of cDNA using primersbased on the DNA sequence in FIG. 1. PCR methods are described in, forexample, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold SpringHarbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCR Technology,(Stockton Press, N.Y., 1989).

The construction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning - - - ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (most recentSupplement)).

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used, the term nucleotide sequence “coding forexpression of” a polypeptide refers to a sequence that, upontranscription and translation of mRNA, produces the polypeptide. As anyperson skilled in the are recognizes, this includes all degeneratenucleic acid sequences encoding the same amino acid sequence. This caninclude sequences containing, e.g., introns. As used herein, the term“expression control sequences” refers to nucleic acid sequences thatregulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are “operativelylinked” to a nucleic acid sequence when the expression control sequencescontrol and regulate the transcription and, as appropriate, translationof the nucleic acid sequence. Thus, expression control sequences caninclude appropriate promoters, enhancers, transcription terminators, astart codon (i.e., ATG) in front of a protein-encoding gene, splicingsignals for introns, maintenance of the correct reading frame of thatgene to permit proper translation of the mRNA, and stop codons.

The recombinant nucleic acid can be incorporated into an expressionvector comprising expression control sequences operatively linked to therecombinant nucleic acid. The expression vector can be adapted forfunction in prokaryotes or eukaryotes by inclusion of appropriatepromoters, replication sequences, markers, etc.

The recombinant nucleic acid used to transfected the cell containsexpression control sequences operably linked to a nucleotide sequenceencoding a β-lactamase. The β-lactamase encoded can be any known to theart or described herein. This includes, for example, the enzymes shownin FIG. 7.

This invention provides novel recombinant nucleic acid moleculesincluding expression control sequences adapted for function in anon-mammalian eukaryotic cell operably linked to a nucleotide sequencecoding for the expression of a cytosolic β-lactamase. As used herein,“cytosolic β-lactamase” refers to a β-lactamase that lacks amino acidsequences for secretion from the cell membrane, e.g., the signalsequence. For example, in the polypeptide of Sequence 1 of FIG. 7, thesignal sequence has been replaced with the amino acids Met-Ser.Accordingly, upon expression, this β-lactamase remains within the cell.

This invention provides recombinant nucleic acid molecules includingexpression control sequences adapted for function in a mammalianeukaryotic cell operably linked to a nucleotide sequence coding for theexpression of a β-lactamase.

It is further preferable that the ribosome binding site and nucleotidesequence coding for expression of β-lactamase contain sequencespreferred by mammalian cells. Such sequences improve expression ofβ-lactamase in mammalian cells. Preferred sequences for expression inmammalian cells are described in, for example, Kozak, M., J. Cell Biol.108:229-241 (1989), referred to herein as “Kozak sequences”. Thenucleotide sequence for cytosolic β-lactamase in Sequence 3 of FIG. 7contains Kozak sequences for the nucleotides −9 to 4 (GGTACCACCATGA).

When used in mammalian cells, the expression control sequences areadapted for function in mammalian cells. The method of this invention isuseful to testing expression from any desired set of expression controlsequences. In particular, this invention is useful for testingexpression from inducible expression control sequences. As used herein,“inducible expression control sequences” refers to expression controlsequences which respond to biochemical signals either by increasing ordecreasing the expression of sequences to which they are operablylinked. For example, in the case of genes induced by steroid hormones,the expression control sequences includes hormone response elements. Thebinding of a steroid hormone receptor to the response element inducestranscription of the gene operably linked to these expression controlsequences. Expression control sequences for many genes and for induciblegenes, in particular, have been isolated and are well known in the art.The invention also is useful with constitutively active expressioncontrol sequences.

The transfected cell is incubated under conditions to be tested forexpression of β-lactamase from the expression control sequences. Thecell or an extract of the cell is contacted with a β-lactamase substrateof the invention under selected test conditions and for a period of timeto allow catalysis of the substrate by any β-lactamase expressed. Thenthe donor moiety from this sample is excited with appropriateultraviolet or visible wavelengths. The degree of fluorescence resonanceenergy transfer in the sample is measured.

If the cell did not express β-lactamase, very little of the substratewill have been cleaved, the efficiency of FRET in the cell will be high,and the fluorescence characteristics of the cell or sample from it willreflect this efficiency. If the cell expressed a large amount ofβ-lactamase, most of the substrate will be cleaved. In this case, theefficiency of FRET is low, reflecting a large amount or high efficiencyof the cleavage enzyme relative to the rate of synthesis of the tandemfluorescent protein construct. In one aspect, this method can be used tocompare mutant cells to identify which ones possess greater or lessenzymatic activity. Such cells can be sorted by a fluorescent cellsorter based on fluorescence.

Also, as will be apparent to those working in the field of usingreporter gene cell-based assays for screening samples or pools ofsamples (such as compounds (combinatorial or synthetic), natural productextracts, or marine animal extracts) to identify potential drugcandidates which act as agonists, inverse agonists or antagonists ofcellular signaling or activation, the combination of cells (preferablymammalian) genetically engineered to express beta-lactamase under thecontrol of different regulatory elements/promoters and the use of thenovel beta-lactamase substrate compounds of the present invention willprovide distinct advantages over known reporter genes (including, butnot limited to, chloramphenicol acetyl transferase, firefly luciferase,bacterial luciferase, Vargula luciferase, aequorin, beta-galactosidase,alkaline phosphatase) and their requisite substrates.

By the choice of appropriate regulatory elements and promoters tocontrol expression of beta-lactamase, assays can be constructed todetect or measure the ability of test substances to evoke or inhibitfunctional responses of intracellular hormone receptors. These includeexpression control sequences responsive to inducible bymineralcorticosteroids, including dexamethasone [J. Steroid Biochem,Molec. Biol. Vol. 49, No. 1 1994, pp.31-3]), gluococorticoid, andthyroid hormone receptors [as described in U.S. Pat. No. 5,071,773].Additional such intracellular receptors include retinoids, vitamin D3and vitamin A [Leukemia vol 8, Suppl. 3, 1994 ppS1-S10; Nature Vol. 374,1995, p.118-119; Seminars in Cell Biol., Vol. 5, 1994, p.95-103].Specificity would be enabled by use of the appropriate promoter/enhancerelement. Additionally, by choice of other regulatory elements orspecific promoters, drugs which influence expression of specific genescan be identified. Such drugs could act on specific signaling moleculessuch as kinases, transcription factors, or molecules such signaltransducers and activators of transcription [Science Vol 264, 1994,p.1415-1421; Mol. Cell Biol., Vol. 16, 1996, p.369-375]. Specificmicrobial or viral promoters which are potential drug targets can alsobe assayed in such test systems.

Also by the choice of promoters such as c-fos or c-jun [U.S. Pat. No.5,436,128; Proc. Natl. Acad. Sci. Vol. 88, 1991, pp. 5665-5669] orpromoter constructs containing regulatory elements responsive to secondmessengers [Oncogene, 6:745-751 (1991)] (including cyclic AMP-responsiveelements, phorbol ester response element (responsive to protein kinase Cactivation), serum response element (responsive to protein kinaseC-dependent and independent pathways) and Nuclear Factor of ActivatedT-cells response element (responsive to calcium) to control expressionof beta-lactamase, assays can be constructed to detect or measuresubstances or mixtures of substances that modulate cell-surfacereceptors including, but not limited to, the following classes:receptors of the cytokine superfamily such as erthyropoietin, growthhormone, interferons, and interleukins (other than IL-8) andcolony-stimulating factors; G-protein coupled receptors [U.S. Pat. No.5,436,128] for hormones, such as calcitonin, epinephrine or gastrin,pancrine or autocrine mediators, such as stomatostatin orprostaglandins, and neurotransmitters such as norepinephrine, dopamine,serotonin or acetylcholine; tyrosine kinase receptors such as insulingrowth factor, nerve growth factor [U.S. Pat. No. 5,436,128].Furthermore, assays can be constructed to identify substances thatmodulate the activity of voltage-gated or ligand-gated ion channels,modulation of which alters the cellular concentration of secondmessengers, particularly calcium [U.S. Pat. No. 5,436,128]. Assays canbe constructed using cells that intrinsically express the promoter,receptor or ion channel of interest or into which the appropriateprotein has been genetically engineered.

The expression control sequences also can be those responsive tosubstances that modulate cell-surface receptors or the modulateintra-cellular receptors.

To measure whether a substance or mixture of substances activatesextracellular or intracellular receptors or other cellular responses,cells containing beta-lactamase controlled by a desiredpromoter/enhancer element are incubated with test substance(s),substrate then added, and after a certain period of time thefluorescence signal is measured at either one or two excitation-emissionpairs appropriate to the chosen compound of the invention (e.g. compoundCCF2 with wavelength pairs of near 405 nm and near 450 nm and near 405and near 510 nm). This fluorescent result is compared to control sampleswhich have had no drug treatment and, when feasible, control sampleswith a known inhibitor and a known activator. The effect of any activedrugs is then determined using the ratio of the fluorescence signalfound in test wells to the signals found in wells with no drugtreatment. Assays are performed in wells in a microtiter platecontaining 96 or more wells or in an assay system with no compartmentssuch as a gel matrix or moist membrane environment. Detection could bedone for example by microtiter plate fluorimeters, e.g. MilliporeCytofluor, or imaging devices capable of analyzing one or more wells orone or more assay points in a certain surface area, e.g. as supplied byAstromed. The ability to retain the substrate in the cytoplasm of livingcells is advantageous as it can allow a reduction in signal interferencefrom coloured or quenching substances in the assay medium. Furthermore,the fluorescent signal from he compounds of this invention, such asCCF2, can be readily detected in single cells and thus allowing assayminiaturization and an increased number of tests per surface area.Miniaturized assays also further increase the throughput of an imagingdetection system as there are more samples within the imaging field.

The assay systems of the present invention further provide anadvantageous and rapid method of isolation and clonal selection ofstably transfected cell lines containing reporter genes and having thedesired properties which the transfection was intended to confer, e.g.fluorescent signal response after activation of a transfected receptorwith a high signal-to-noise ratio of at least 10:1 from a highproportion of isolated cells. Current procedures for clonal selection ofsatisfactorily transfected, genetically engineered cells from thepopulation initial transfected with the vectors of interest, are donemainly by manual means and involve several rounds of microscopicanalyses, selecting the visually preferred clone, isolation of the cloneby manual pipetting stages and prolonged cellular cultivations. Thisprocedure is laborious and time-consuming; it may require several monthsto generate a clone useful for assays suited to drug screening.Moreover, it is difficult to manually select and maintain more than afew hundred clones. Using the assays of this present invention, thedesired signal from cellular beta-lactamase reporter system can bemaintained within living and viable cells. Thus, one can rapidly select,from the population of initially transfected cells, those few livingcells with the best fluorescent signal using automated instruments suchas a fluorescent-activated cell sorter, e.g. the Becton Dickinson FACSVantage. The selected cells are then collected for cultivation andpropagation to produce a clonal cell line with the desired propertiesfor assays and drug screening.

in addition, the presence (for example, in human serum, pus or urine) ofbacteria resistant to β-lactam antibiotics can be readily detected usingthe substrates of the present invention. Only in the presence of anactive β-lactamase is there a change in the fluorescence spectrum fromthat of the intact molecule to one characteristic of the cleavageproduct. The substrates of the present invention are superior to priorart chromogenic substrates Nitrocephin and PADAC, in that the inventivesubstrates are stable to human serum. The novel substrates are also moresensitive than the chromogenic substrate CENTA, because they experiencea much smaller optical background signal from human serum and a lowerdetection limit for fluorescence versus absorbance.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should be not be construed as in any sense limiting the scopeof the invention as defined in the claims appended hereto.

MEASUREMENTS

The degree of FRET can be determined by an spectral or fluorescencelifetime characteristic of the excited construct, for example, bydetermining the intensity of the fluorescent signal from the donor, theintensity of fluorescent signal from the acceptor, the ratio of thefluorescence amplitudes near the acceptor's emission maxima to thefluorescence amplitudes near the donor's emission maximum, or theexcited state lifetime of the donor. For example, cleavage of the linkerincreases the intensity of fluorescence from the donor, decreases theintensity of fluorescence from the acceptor, decreases the ratio offluorescence amplitudes from the acceptor to that from the donor, andincreases the excited state lifetime of the donor.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor moities, a process referred to as “ratioing.” Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore the ratio of the two emissionintensities is a more robust and preferred measure of cleavage thaneither intensity alone.

The excitation state lifetime of the donor moiety is, likewise,independent of the absolute amount of substrate, excitation intensity,or turbidity of other background absorbances. Its measurement requiresequipment with nanosecond time resolution, except in the special case oflanthanide complexes in which case microsecond to millisecond resolutionis sufficient.

Additional suitable Chel moieties are described in Vallarino, L. M., &Leif. R. C., U.S. Pat. No. 5,373,093; Sabbatini, N. et al, Pure andApplied Chem. 67: 135-140 (1995); Mathis, G., Clinical Chem. 41:1391-1397 (1995); Horiguchi, D., Chem. Pharm. Bull. 42: 972-975 (1994);Takalo, H. et al, Bioconjugate Chem. 5: 278-282 (1994); Saha, A. K. etal, J. Amer. Chem. Soc. 115: 11032 (1993); Li, M. & Selvin, P. R., J.Amer. Chem. Soc. 117: 8132-8138 (1995).

Fluorescence in a sample is measured using a fluorimeter. In general,excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation which has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation state, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

Method of performing assays on fluorescent materials are well known inthe art and are described in, e.g., Lakowica, J. R., Principles ofFluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

EXAMPLES

All silica gel chromatography was performed using silica gel (Merck,grade 60, 230-400 mesh, 60 Å) purchased from Aldrich. BakerbondOctadecyl from J. T. Baker was used for C₁₈ reverse phasechromatography. Solvents (high pressure liquid chromatography grade)were used for chromatography as received, or dried over activatedmolecular sieves (3 Å) for synthetic purposes.

Fluorescence excitation and emission spectra were measured either on aSpex Fluorolog 111 or on a K2fluorometer (ISS, Champaigne, Ill.) inratio mode with a rhodamine B quantum counter. The efficiency offluorescence energy transfer was determined from the change in theintegrated fluorescence emission at the donor emission wavelength upontreatment with β-lactamase. For fluorescence microscopy imaging, twodifferent imaging setups were used. One, with an inverted fluorescencemicroscope, Zeiss IM-35 (Thornwood, N.Y.) coupled to asilicon-intensified target (SIT) camera (Dage-MTI, Michigan City, Ind.)has been described in detail [Tsien, R. Y. (1986) New tetracarboxylatechelators for fluorescence measurement and photochemical manipulation ofcytosolic free calcium concentrations, in: Optical Methods in CellPhysiology, ed. de Weer, P & Salzberg, B., New York:Wiley, pp. 327-345;Tsien and Harootunian (1990) Cell Calcium 11:93 -109]. The otherconsisted of a cooled charge-coupled-device (CCD) camera (Photometrics,Tucson, Ariz.) connected to an inverted fluorescence microscope (ZeissAxiovert).

Fluorescence resonance energy transfer was measured by monitoring theratio of fluorescence intensities at donor and acceptor emissionwavelengths using commercially-available filters (Omega Optical).

Excitation: 360 DF 40, dichroic mirror 390 DCLP or 405 DF 15, dichroicmirror 420 DRLPO2 Emission: 450 DF 65 (donor emission) 515 EFLP(acceptor emission) 435 EFLP (to view donor and acceptor fluorescencesimultaneously)

Example 1 (Compound 11)

Test the optical properties of a cephalosporin with two dye moleculesattached the following model compound was synthesized.

The first synthesis step was to convert 7-aminocephalosporanic acid intoa bifunctional cephalosporin carrying a thiol in the 3′- position andthe 7-amine [Van Heyningen, E. and Brown, C. N., J. Med. Chem. 8:174-181 (1965); Japanese Patent, Kokai 75/18494, CA 85, 97320d]. Thiscephalosporin was then reacted selectively with an tiol-reactive dye,followed by an amine-reactive dye. The thiol-reactive dye5,(6)-iodoacetamido-fluorescein and the amine-reactive dye5,(6)-carboxy-N,N,N′,N′-tetramethylrhodamine-succinimide were coupled tothe cephalosporin in aqueous dimethylformamide at pH 8. The product willbe referred to as RCF.

In phosphate buffer at pH 7 RCF is virtually non fluorescent; neitherfluorescein nor rhodamine show much fluorescence when excited at theirrespective excitation maxima, which is indicative of chromophorestacking (“dark complex”). After long term treatment with β-lactamasethe β-lactam is cleaved causing the fluorescence of both dyes toreappear (FIGS. 1(a) and 1(b)). This experiment confirms that one canmeasure β-lactamase catalyzed hydrolysis of the β-lactam incephalosporins by the loss of fluorescence quenching using anappropriate donor-acceptor pair.

Example 2

The thiomethyl linker was introduced by conversion of5-fluoresceninamine to 5-mercaptofluorescein via diazotization,conversion of the ethylxanthate, and degradation of the xanthate byaqueous acid to the free sulfhydryl. It was coupled to7-bromoacetamido-cephalosporanic acid by necleophilic displacement ofthe bromide by the mercapto group of the fluorescein.7-Bromoacetamido-cephalosporanic acid had been prepared from7-aminocephalosporanic acid and bromoacetyl bromide [Bunnell, C. A. etal. Industrial manufacture of cephalosporins. In: Beta-LactamAntibiotics for Clinical Use. Series: Clinical Pharmacology Vol. 4,edited by Queener, S. F., Webber, J. A. and Queener, S. W. New York: M.Dekker, 1986, p. 255-283].

To prepare 7β-[(5-diacetylfluorescein)thio]acetamido-3-(acetoxymethyl)-3-cephem-4-carboxylicacid (14), in a nitrogen atmosphere 130 mg (0.29 mmol)5-fluoresceinthiol diacetate were dissolved in 10 ml dimethylformamideand added to 120 mg (0.31 mmol)7β-bromoacetamido-3-(acetoxymethyl)-3-cephem-4-carboxylic acid in 10 ml1M potassium phosphate buffer adjusted to pH 8.0. The solution wasstirred for 8 hours at room temperature after which the solvents wereremoved in vacuo. The residue was dissolved in 10 ml water and the pH ofthe solution was carefully adjusted to pH 5 with dilute phosphoric acid.At this point nonpolar byproducts precipitated and were removed bycentrifugation. Further acidification to pH 2.7 precipitated the titlecompound which was collected by centrifugation, washed 3 times with 2 mldiethylether-tetrachloromethane (1:2), and dried in vacuo. ¹H NMR(CDCl₃): δ2.08 ppm (s, 3H, acetate), δ3.36 ppm, 3.53 ppm (2d, 2H, J=17.3Hz, C-2), δ3.87 ppm (s, 2H, side chain methylene), δ4.88 ppm, 5.16 ppm(2d, 2H, J=13.6 Hz, C-3′), δ4.96 ppm (d, 1H, J=4.9 Hz, C-6), δ5.81 ppm(dd, 1H, J₁=8.2 Hz, J₂=4.9 Hz, C-7), δ6.85 ppm (m, 4H, xanthene), δ7.10(s, 2H, xanthene), δ7.15 ppm (d, 1H, J=8.2 Hz, amide), δ7.69 ppm (d, 1H,J=8.2 Hz, phthalic), δ7.91 ppm (d, 1H, J=8.2 Hz, phthalic), δ8.11 ppm(s, 1H, phthalic).

5-Fluoresceinamine was brominated to generate 5-eosinamine, which wasconverted into 5-mercaptoeosin in analogous way to the5mercaptofluorescein. In a nucleophilic displacement of thecephalosporin acetate by 5-mercaptoeosin diacetate theFRET-cephalosporin was generated as the protected tetraacetylderivative.

To prepare 5-eosinamine, 1.74 g (5 mmol) 5-fluoresceinamine wassuspended in 30 ml glacial acetic and 2.06 ml (40 mmol, 100% excess)bromine was added. With the addition of bromine the fluoresceinaminewent into solution. The solution was heated for six hours at 90° C.,during which period a white precipitate began to form. An ice-cooledtrap attached to the flask kept bromine from escaping from theatmosphere. Excess bromine was then recovered by distillation into aliquid nitrogen cooled collecting flask. One volume of water was addedto the acetic acid solution to precipitate any product remaining insolution. The precipitate was collected by filtration and dissolved in1N aqueous sodium hydroxide. 5-Eosinamine was precipitated as the freeamine by addition of glacial acetic acid. The eosinamine was dissolvedin little chloroform and methanol was added. Concentrating this solutionon the rotary evaporator gave 2.56 g (3.85 mmol, 77%) eosinamine as afine white powder (the eosinamine-spirolactone).

To prepare 5-eosin-ethylxanthate diacetate, 670 mg (1mmol) 5-eosinaminewere stirred in 2 ml concentrated sulfuric acid and 2 ml glacial aceticacid. The suspension was cooled with an ice-salt bath to a few degreesbelow 0° C., which turned it into a thick paste that was difficult tostir. 200 mg (2.9 mmol) sodium nitrite in 1 ml water were added dropwiseover the period of one hour. After another 2 hours at 0° C. 20 g of icewas slowly added. The flask was put on the high vacuum pump in the cold,to remove excess nitrous gases (caution!!). Saturated ice-cold aqueoussodium bicarbonate solution was added until the solids dissolved intothe dark red solution. 200 mg (1.2 mmol) Potassium ethylxanthate wasadded and a pink precipitate formed (5-eosindiazonium xanthate). A fewcrystals of nickel(II)chloride catalyzed the conversion of the diazoniumsalt with evolution of nitrogen. Once nitrogen evolution had ceased theproducts were precipitated with 1N hydrochloric acid. The precipitatewas collected by filtration and dried in vacuo. It was treated withacetic anhydride-pyridine (1:1) at 40° C. for one hour. After removal ofthe reagents in vacuo, the residue was chromatographed over silica gelwith ethyl acetate-hexane (1:4) as eluent. The desired product elutedfirst. The yield was 110 mg (0.13 mmol, 13%) of the title compound as awhite powder.

For preparation of the disulfide dimer of 5-eosinthiol diacetate (dimerof 15), 110 mg (0.13 mmol) 5-eosin-ethylxanthate diacetate was stirredin 10 ml concentrated (3%) aqueous ammonia and the solution was heatedto 70° C. Air was bubbled slowly through the solution to oxidize thethiol to the disulfide in situ. After 2 hours the solvents were removedon the rotary evaporator at 40° C. and the residue was treated withacetic anhydride-pyridine (1:1). After removal of the reagents in vacuothe residue was chromatographed over silica gel with ethylacetate-hexane (1:4) as the eluent. Yield was 90 mg (60 μmol, 91%) ofthe title product as a white powder. The compound was reduced to themonomer (15) by dissolving it in methanol with addition of sodiumacetate and addition of 20 equivalents mercaptoethanol. After 2 hoursthe methanolic solution was poured into 3 volumes 5% aqueous acetic acidfrom which the precipitating 5-fluoresceinthiol monomer was collected bycentrifugation. The solid was washed with water until no odor ofmercaptoethanol remained.

Coupling of diacetyl 5-eosinthiol (15) with7β-[(5-diacetylfluorescein)thio]acetamido-3-(acetoxymethyl)-3-cephem-4-carboxylicacid (14) and deacylation with acetylesterase was effected as follows.10 mg (13 μmol)7β-[(5-Diacetylfluorescein)thio]acetamido-3-(acetoxymethyl)-3-cephem-4-carboxylicacid and 10 mg (13 μmol) diacetyl-5-eosinthiol were dissolved in 200 μldry acetonitrile and the solution was sealed under argon in a glasstube. The tube was kept in an oil bath at 84° C. (±2° C.) for 16 hours.Then it was cut open, the solution transferred to a flask and thesolvent removed in vacuo. The residue was flash-chromatographed oversilica gel with ethyl acetate-methanol-acetic acid (100:1:1) as theeluent. Deprotection of the acetates was achieved by incubating theproduct with orange peel acetylesterase in 50 mM phosphate buffer (pH7)for 24 hours at 37° C. The deacylated product was purified by C₁₈reverse phase chromatography. The eluent was a step gradient of 25 mMaqueous phosphate buffer (pH7) and methanol. Fluorescein byproductseluted with 33% and 50% methanol in the eluent, after which the desiredproduct eluted in 66% methanol.

The deprotected compound shows little fluorescence in phosphate bufferas the two hydrophobic dyes stack. The remaining fluorescence is due tofluorescence resonance energy transfer (FRET). This compound is a goodsubstrate for RTEM β-lactamase and will be referred to as FCE.

Cleavage of the compound increases fluorescence at 515 nm about 70-fold(FIG. 2). The fluorescence properties of the compound can be attributedto dye-dimer formation, as FRET increases drastically once methanol isadded to the solution. Methanol breaks the hydrophobic interaction thatcauses the fluorophores to stack.

Example 3 (compound 22)

The 3′-acetate of 7-aminocephalosporanic acid was displaced byethylxanthate [Van Heyningen and Brown (1965), supra] which washydrolysed to the free sulfhydryl with aqueous acetylhydrazine [JapanesePatent, Kokai 75/18494, CA85, 97320d]. The sulfhydryl group was reactedwith 5-bromoacetamido-rhodol-X in aqueous dimethylformamide. Thecephalosporin 7 amine was reacted with btromacetyl bromide in aqueousdioxane, followed by bromide displacement with 5-fluoresceinthiol toyield a FRET-cephalosporin that is virtually nonfluorescent in 50 mMphosphate buffer pH 7. This compound is referred to as FCRX.

The first step in preparation of 5-rhodol-X-bromacetamide was synthesisof 9-(2′-carboxy-4′(5′)-nitro-benzoyl)-8-hydroxyjulolidine andseparation of the isomers. 10.1 g (48 mmol, 92% purity) 4-Nitrophthalicanhydride were dissolved in 20 ml toluene at 7° C. 97.6 g (50 mmol, 97%purity) 8-Hydroxyjulolidine in 20 ml ethyl acetate were added and thesolution kept at 70° C. for 30 min. The reaction mixture was run througha short bed of silica gel followed by ethyl acetate as eluent. Thesolvents were removed in vacuo and the solid redissolved in a minimumamount of refluxing ethyl acetate. The isomer with the nitro-group metato the benzoic acid crystallizes over night from solution in orangecrystals (3.47 g in first fraction). After additional fractionalcrystallization the pure isomer was obtained. ¹H NMR (CDCl₃) ofcrystallized isomer: δ1.91 ppm (m, 4H, aliphatic methylenes), δ2.73 ppm,2.46 ppm (2m, 4H, anilinic methylenes), δ3.26 ppm (m, 4H, benzylicmethylenes), δ6.32 ppm (s, 1H, julolidine), δ7.53 ppm (d, 1H, J=8.4 Hz,phthalic), δ8.43 ppm (dd, J₁=8.4 Hz, J₂=2.2 Hz, phthalic), δ8.90 ppm (d,1H, J=2.2 Hz, phthalic).

For preparation of 5rhodol-X-amine hydrochloride (named by analogy withrhodamine-X), 1.91 g (5.0 mmol) 9-(2′-nitro-benzoyl)-8-hydroxyjulolidinewas stirred in 5 ml concentrated (96%) sulfuric acid. 700 mg (6.4 mmol,1.25 equ.) resorcinol was added with cooling over a period of 15minutes. The suspension was stirred 1.5 hours at room temperature andthen poured into 200 ml water with vigorous stirring. The purpleprecipitate was collected by filtration and redissolved in 75 ml waterwith the help of 5.3 g (22 mmol) sodium sulfide nonahydrate. 2.5 g (44.6mmol) Anhydrous sodium bisulfide was added and the solution refluxed for24 hours. Then, after cooling to room temperature, the product wasprecipitated by addition of glacial acetic acid. The solid was collectedby filtration and boiled with 100 ml half-saturated aqueous hydrochloricacid. The solution was filtered hot through a glass frit to removesulfur. The solution volume was reduced to 10 ml on the rotaryevaporator. 1 Volume saturated brine was added and the precipitatecollected by filtration. Crystallization from refluxing hydrochloricacid yielded 1.78 g (3.85 mmol, 77%) dark red crystals of5-rhodol-X-amine hydrochloride. ¹H NMR (dDMSO) of 5-nitro-rhodol-X:δ1.90 ppm, 2.05 ppm (2m, 4H, aliphatic methylenes), δ2.72 ppm, 3.03 ppm(2m, 4H, anilinic methylenes), δ3.66 ppm (m, 4H benzylic methylenes),δ6.90 ppm (s, 1H, xanthene), δ6.96 ppm (dd, 1H, J₁=9.0 Hz, J₂=2.1 Hz,xanthene), δ7.11 ppm (d, 1H, J=9.0 Hz, xanthene), δ7.22 ppm (d, 1H,J=2.1 Hz, xanthene), δ7.78 ppm (d, 1H, J=8.4 Hz, phthalic), δ8.70 ppm(dd, 1H, J₁=8.4 Hz, J₂=2.4 Hz, phthalic), δ8.91 ppm (d, 1H, J=2.4 Hz,phthalic). ¹NMR (CD₃OD) of 5-rhodol-X-amine hydrochloride: δ2.00 ppm,2.14 ppm (2m, 4H, aliphatic methylenes), δ2.75 ppm, 3.11 ppm (2m, 4H,anilinic methylenes), δ3.67 ppm (m, 4H, benzylic methylenes), δ6.85 ppm(s, 1H, xanthene), δ6.94 ppm (dd, 1H, J₁=9.0 Hz, J₂=2.1 Hz, xanthene),δ7.13 ppm (d, 1H, J=9.0 Hz, xanthene), δ7.16 ppm (d, 1H, J=2.1 Hz,xanthene), δ7.55 ppm (d, 1H, J=8.1 Hz, phthalic), δ7.82 pm (dd, 1H,J₁=8.1 Hz, J₂=1.9 Hz, phthalic), δ8.28 pm (d, 1H, J=1.9 Hz, phthalic).

Preparation of 5-rhodol-X-bromoacetamide (18) was effected as follows.115 mg (0.25 mmol) 5-Rhodol-X-amine hydrochloride were dissolved with180 mg (2.1 mmol) sodium bicarbonate in 2 ml water-dioxane (1:1). Thesolution was cooled on ice and 175 μl (2 mmol) bromoacetylbromide wereadded with stirring over a period of 20 minutes. The solution was thenkept at room temperature for 1.5 hours, after which 5 volumes of waterwere added. The dioxane was removed on the rotary evaporator, and theproduct was precipitated from the remaining aqueous solution by additionof acetic acid. The precipitate was filtered off and dissolved in asmall volume of chloroform-methanol (1:1). Silica gel was added to thesolution and the solvents removed in vacuo. The solids were applied to asilica gel column and the product eluted with methanol-ethyl acetate(1:4). This eluent did dissolve some silica gel which remained with theeluted product. ¹H NMR (CD₃OD, 10% dDMSO): δ1.98 ppm, 2.12 ppm (2m, 4H,aliphatic methylenes), δ2.72 ppm, 3.06 ppm (2m, 4H, anilinicmethylenes), δ3.56 ppm (m, 4H, benzylic methylenes), δ4.08 ppm (s, 2H,bromoacetyl), δ6.79 ppm (dd, 1H, J₁=9.2 Hz, J₂=2.1 Hz, xanthene), δ6.83ppm (s, 1H, xanthene), δ6.90 ppm (d, 1H, J=2.1 Hz, xanthene), δ7.19 ppm(d, 1H, J=9.2 Hz, xanthene), δ7.24 ppm (d, 1H, J=8.4 Hz, phthalic),δ8.02 ppm (dd, 1H, J₁=8.4 Hz, J₂˜1 Hz, phthalic), δ8.30 ppm (d, 1H, J˜1Hz, phthalic).

For preparation of7β-(bromoacetamido)-3-[[[(5-rhodol-X-amido)methyl]thio]methyl]-3-cephem-4-carboxylicacid (2), 4.5 mg (10 μmol) 5-Rhodol-X-bromoacetamide (18) were dissolvedin 0.5 ml 250 mM phosphate buffer adjusted to pH 7.7 and 0.5 mldimethylformamide. The solution was deoxygenated and 10 mg (40 μmol)7β-amino-3-(thiomethyl)-3-cephem-4-carboxylic acid (8) preparedaccording to the literature procedure in 100 μl phosphate buffer wasadded in an argon atmosphere. The solution was kept for 2 hours at 30°C. Then the solvents were removed in vacuo and the residue dissolved in1 ml water, from which the product was precipitated by addition ofacetic acid. The precipitate was collected and the product purified byC₁₈ reverse-phase chromatography with 0.1% trifluoroacetic acid in 35%methanol/water as eluent.

The above product (19) was dissolved in 1 ml dioxane-water (1:1) with 20mg sodium bicarbonate. 10 μl Bromoacetyl bromide were added to thesolution on ice. The solution was kept for another 1.5 hours at roomtemperature. 20 mg sodium bicarbonate and 10 μl bromoacetyl bromide wereadded to the solution with ice cooling. After another 1.5 hours at roomtemperature the dioxane was removed on the rotary evaporator and theproducts were precipitated from the aqueous solution with 1M phosphoricacid and collected by centrifugation. The solids were suspended indilute aqueous bicarbonate solution and the undissolved particlesremoved by centrifugation and discarded. The product was precipitatedwith 1M phosphoric acid and purified by flash chromatography on silicagel with chloroform-methanol-acetic acid-water (55:15:4:2). Thisprocedure dissolved small amounts of silica gel.

Coupling of diacetyl 5-fluoroesceinthiol (21) with7β-(bromoacetamido)-3-[[[(5-rhodol-X-amido)methyl]thio]methyl]-3-cephem-4-carboxylicacid (20) was effected as follows.7β-(Bromoacetamido)-3-[[[(5-rhodol-X-amido)methyl]thio]methyl]-3-cephem-4-carboxylicacid was reacted with a 50% excess of 5-fluoresceinthiol under argonwith dimethylformamide-(250 mM aqueous phosphate buffer pH 7.7) (1:1) asthe solvent. The product was purified from excess fluoresceinthiol byrepeated dissolution in methanol and precipitation in ethyl acetate.

FIG. 3 shows the fluorescence emission spectra of thisFRET-cephalosporin in 50 mM phosphate buffer pH 7 before and aftertreatment with β-lactamase. The low initial fluorescence is due to thestacking of the fluorophores, forming a ground state complex that isnonfluorescent. When one adds methanol to the solution this stacking canbe disrupted and efficient fluorescence resonance energy transferoccurs.

Example 4 (compound 25)

N-[resorufin-4-carbonyl]-N′-iodoacetyl-piperazine (Boehringer Mannheim)was attached to the cephalosporin as a FRET-acceptor for fluorescein. Itis referred to as FCRE.

The FRET-cephalosporin FCRE (25) carrying fluorescein as the donor andresorufin as the quencher was made by the same procedure as the onecarrying the rhodol-X-acceptor. TheN-[resorufin-4-carbonyl]-N′-iodoacetyl-piperazine (Boehringer Mannheim)was coupled to the free 3′-thiol of the cephalosporin followed bybromoacetylation and addition of the 5-fluoresceinthiol. In departurefrom the protocol, three equivalents of 5-fluorescein thiol were added,as the first equivalent instantaneously reduced the resorufin and formedunreactive difluorescein-disulfide. Exposure to air reoxidized resorufinto the original dye.

β-Lactamase catalyzed hydrolysis of this compound generates twofluorescent fragments. Resorufin excitation and emission spectra arelonger wavelength and narrower than the rhodol spectra, possiblyaffording better spectral separation between the uncleaved dye versusthe products of enzymatic cleavage. But, as in the case of rhodol as theacceptor, in aqueous phosphate buffer the dyes stack and form a darkcomplex. β-Lactamase treatment disrupts the stacking and increases donorfluorescence (FIG. 4).

Example 5 (compound 7b)

For synthesis of 2,4 dihydroxy-5-chlorobenzaldehyde, 21.7 g (0.15 Mol)4-chlororesorcinol were dissolved in 150 ml dry diethyl ether and 27 gfinely powdered zinc (II) cyanide and 0.5 g potassium chloride wereadded with stirring. The suspension was cooled on ice. A strong streamof hydrogen chloride gas was blown into the solution with vigorousstirring. After approximately 30 minutes the reactants were dissolved.The addition of hydrogen chloride gas was continued until it stoppedbeing absorbed in the ether solution (approx. 1 hour). During this timea precipitate formed. The suspension was stirred for one additional houron ice. Then the solid was let to settle. The ethereal solution waspoured from the solid. The solid was treated with 100 g of ice andheated to 100° C. in a water bath. Upon cooling the product crystallizedin shiny plates from the solution. They were removed by filtration ondried over potassium hydroxide. The yield was 15.9 g (0.092 Mol, 61%).¹H NMR (CDCl₃): δ6.23 ppm (s, 1H, phenol), δ6.62 ppm (s, 1H, phenyl),δ7.52 ppm (s, 1H, phenyl), δ9.69 ppm (s, 1H, formyl), δ11.25 ppm (s, 1H,phenol).

To prepare 3-carboxy 6-chloro 7-hydroxy coumarin, 5.76 g (0.033 Mol)2,4-dihydroxy-5-chlorobenzaldehyde and 7.2 g (0.069 Mol) malonic acidwere dissolved in 5 ml warm pyridine. 75 μl Aniline were stirred intothe solution and the reaction let to stand at room temperature for3days. The yellow solid that formed was broken into smaller pieces and50 ml ethanol was added. The creamy suspension was filtered through aglass frit and the solid was washed three times with 1 N hydrochloricacid and then with water. Then the solid was stirred with 100 ml ethylacetate, 150 ml ethanol and 10 ml half concentrated hydrochloric acid.The solvent volume was reduced in vacuo and the precipitate recovered byfiltration, washed with diethyl ether and dried over phosphorouspentoxide. 4.97 g (0.021 Mol, 63%) of product was obtained as a whitepowder. ¹H NMR (dDMSO): δ6.95 ppm (s, 1H), δ8.02 ppm (s, 1H), δ8.67 ppm(s, 1H).

To prepare 7-butyryloxy-3-carboxy-6-chlorocoumarin, 3.1 g (12.9 mMol)3-carboxy-6-chloro-7-hydroxycoumarin were dissolved in 100 ml dioxaneand treated with 5 ml butyric anhydride, 8 ml pyridine and 20 mgdimethyl aminopyridine at room temperature for two hours. The reactionsolution was added with stirring to 300 ml heptane upon which a whiteprecipitate formed. It was recovered by filtration and dissolved in 150ml ethyl acetate. Undissolved material was removed by filtration and thefiltrate extracted twice with 50 ml 1 N hydrochloric acid/brine (1:1)and the brine. The solution was dried over anhydrous sodium sulfate.Evaporation in vacuo yielded 2.63 g (8.47 mMol, 66%) of product. ¹H NMR(CDCl₃): δ1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl), δ1.85 ppm (m, 2H,J₁˜J₂=7.4 Hz, butyric methylene), δ2.68 ppm (t, 2H, J=7.4 Hz, butyricmethylene), δ7.37 ppm (s, 1H, coumarin), δ7.84 ppm (s, 1H, coumarin),δ8.86 ppm (s, 1H, coumarin).

Preparation of7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarin iseffected as follows. 2.5 g (8.06 mMol)7-Butyryloxy-3-carboxy-6-chlorocoumarin, 2.36 g hydroxybenztriazolehydrate (16 mMol) and 1.67 g (8.1 mMol) dicyclohexyl carbodiimide weredissolved in 30 ml dioxane. A toluene solution of O-benzylglycine[prepared by extraction of 3.4 g (10 mMol) benzylglycine tosyl salt withethyl acetate—toluene—saturated aqueous bicarbonate—water (1:1:1:1, 250ml), drying of the organic phase with anhydrous sodium sulfate andreduction of the solvent volume to 5 ml] was added dropwise to thecoumarin solution. The reaction was kept at room temperature for 20hours after which the precipitate was removed by filtration and washedextensively with ethylacetate and acetone. The combined solventfractions were reduced to 50 ml on the rotatory evaporator upon whichone volume of toluene was added and the volume further reduced to 30 ml.The precipitating product was recovered by filtration and dissolved in200 ml chloroform—absolute ethanol (1:1). The solution was reduced to 50ml on the rotatory evaporator and the product filtered off and dried invacuo yielding 1.29 g of the title product. Further reduction of thesolvent volume yielded a second crop (0.64 g). Total yield: 1.93 g (4.22mMol, 52%). ¹H NMR (CDCl₃): δ 1.08ppm, (t, 3H, J=7.4 Hz, butyricmethyl), δ 1.84ppm (m, 2H, J₁≈J₂=7.4 Hz, butyric methylene), δ 2.66ppm(t, 2H, J=7.4 Hz, butyric methylene), δ 4.29ppm (d, 2H, J=5.5 Hz,glycine methylene), δ 5.24ppm (s, 2H, benzyl), δ 7.36ppm (s, 1H,coumarin), δ 7.38ppm (s, 5H, phenyl), δ 7.77ppm (s, 1H, coumarin), δ8.83ppm (s, 1H, coumarin), δ 9.15ppm (t, 1H, J=5.5 Hz, amide).

7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin was preparedas follows. 920 mg (2 mMol)7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarinwere dissolved in 50 ml dioxane. 100 mg palladium on carbon (10%) and100 μl acetic acid were added to the solution and the suspension stirredvigorously in a hydrogen atmosphere at ambient pressure. After theuptake of hydrogen seized the suspension was filtered. The productcontaining carbon was extracted five times with 25 ml boiling dioxane.The combined dioxane solutions were let to cool upon which the productprecipitated as a white powder. Reduction of the solvent to 20 mlprecipitates more product. The remaining dioxane solution is heated toboiling and heptane is added until the solution becomes cloudy. Theweights of the dried powders were 245 mg, 389 mg and 58 mg, totaling 692mg (1.88 mMol, 94%) of white product. ¹H NMR (dDMSO): δ 1.02ppm (t, 3H,J=7.4 Hz, butyric methyl), δ 1.73ppm (m, 2H, J₁≈J₂=7.3 Hz, butyricmethylene), δ 2.70ppm (t, 2H, J=7.2 Hz, butyric methylene), δ 4.07ppm(d, 2H, J=5.6 Hz, glycine methylene), δ 7.67ppm (s, 1H, coumarin), δ8.35ppm (s, 1H, coumarin), δ 8.90ppm (s, 1H, coumarin), δ 9.00ppm, (t,1H, J=5.6 Hz, amide).

Coupling of 7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarinwith 7-amino-3′-chlorocephalosporanic acid benzhydryl ester was effectedas follows. 368 mg (1 mMol)7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin, 270 mghydroxybenztriazole hydrate and 415 mg (1 mMol) 7-amino-3′-chlorocephalosporanic acid benzhydryl ester were suspended in 40 mldioxane—acetonitrile (1:1). 260 mg (1.25 mMol) dicyclohexylcarbodiimidein 5 ml acetonitrile were added and the suspension was stirredvigorously for 36 hours. The precipitate was removed by filtration andthe volume of the solution reduced to 20 ml on the rotatory evaporator.50 ml Toluene was added and the volume reduced to 30 ml. With stirring50 ml heptane was added and the suspension chilled on ice. Theprecipitate was recovered by filtration. It was redissolved in 10 mlchloroform and the remaining undissolved solids were filtered off.Addition of 2 volumes of heptane precipitated the title product whichwas collected and dried in vacuo and yielded 468 mg (0.64 mMol, 64%)off-white powder. ¹H NMR (CDCl₃): δ 1.08ppm (t, 3H, J=7.4 Hz, butyricmethyl), δ 1.84ppm (m, 2H, J₁≈J₂=7.4 Hz, butyric methylene), δ 2.66ppm(t, 2H, J=7.4 Hz, butyric methylene), δ 3.54ppm, (2d, 2H, J=18.3 Hz,cephalosporin C-2), δ 4.24ppm (2D, 2H, J=5.8 Hz, cephalosporin 3methylene), δ 4.37ppm (d, 2H, J=3.8 Hz, glycine methylene), δ 5.02ppm(d, 1H, J=4.9 Hz, cephalosporin C-6), δ 5.89ppm (dd, 1H, J₁=9.0 Hz,J₂=5.0 Hz, cephalosporin C-7), δ 6.96ppm (s, 1H, benzhydryl), δ7.30-7.45ppm (m, 12H, phenyl, coumarin, amide), δ 7.79ppm (s, 1H,coumarin), δ 8.84ppm (s, 1H, coumarin), δ 9.28ppm (t, 1H, J=3.7 Hz,amide).

Coupling of the above product with 5-fluoresceinthiol was effected asfollows. 90 mg (0.2 mMol) 5-merceptofluorescein diacetate disulfidedimer were dissolved in 10 ml chloroform and treated with 25 μl tributylphosphine and 25 μl water in an argon atmosphere. The solution was keptfor 2 hours at ambient temperature and was then added to a solution of20 mg sodium bicarbonate, 25 mg sodium iodide and 110 mg (0.15 mMol) ofthe above compound in 10 ml dimethylformamide. After 4 hours thesolvents were removed in vacuo and the residue triturated withdiethylether. The solid was dissolved in ethyl acetate—acetonitrile(1:1). After removal of the solvents the residue was triturated oncemore with diethylether yielding 157 mg (0.13 mMol, 88%) of a creamcolored powder product.

A sample of the above compound was treated with a large access oftrifluoroacetic acid—anisole (1:1) at room temperature for 20 minutes.The reagents are removed in vacuo and the residue triturated with ether.High performance liquid chromatography of the solid in 45% aqueousacetonitrile containing 0.5% acetic acid gives a product in which thebutyrate and the diphenylmethyl esters have been cleaved. It waspurified by high performance liquid chromatography on a reverse phaseC₁₈-column using 45% aqueous acetonitrile containing 5% acetic acid asthe eluent.

Deprotection of the fluorescein acetates in compound 27 was accomplishedwith sodium bicarbonate in methanol (room temperature, 30 minutes) toprovide the fluorescent enzyme substrate CCF2. It was purified by highperformance liquid chromatography on a reverse phase C₁₈-column using35% aqueous acetonitrile containing 0.5% acetic acid as the eluent.

Stirring of compound 27 with excess acetoxymethyl bromide in drylutidine produced the membrane permeable derivative of the substrate(CCF2/ac₂AM₂). It was purified by high performance liquid chromatographyon a reverse phase C₁₈-column using 65% aqueous acetonitrile containing0.5% acetic acid as the eluent. CCF2/ac₂AM₂ is readily converted to CCF2in the cells' cytoplasm.

Unlike in Examples 1-4, the donor and acceptor dyes in substrate CCF2 donot stack. The substrate is fully fluorescent in phosphate buffer andthere is no formation of the “dark complex” (i.e., addition of methanoldoes not change the fluorescence spectrum of CCF2, except for the effectof dilution). This is due to the much smaller and more polar nature ofthe 7-hydroxycoumarin compared to that of the xanthene dyes (eosin,rhodamine, rhodol and resorufin) in Examples 1-4.

FIG. 5 illustrates the emission spectrum of compound CCF2 in 50 mmolarphosphate buffer pH 7.0 before and after β-lactamase cleavage of theβ-lactam ring. In the intact substrate, efficient energy transfer occursfrom the 7-hydroxycoumarin moiety to the fluorescein moiety. Excitationof the substrate at 405 nm results in fluorescence emission at 515 nm(green) from the acceptor due fluorescein. The energy transfer isdisrupted when β-lactamase cleaves the β-lactam ring, thereby severingthe link between the two dyes. Excitation of the products at 405 nm nowresults entirely in donor fluorescence emission at 448 nm (blue). Thefluorescence emission from the donor moiety increases 25 fold uponβ-lactam cleavage. The fluorescence at 515 nm is reduced by 3.5 fold,all of the remaining fluorescence originating from the 7-hydroxycoumarinas its emission spectrum extends into the green. Twenty-five-foldquenching of the donor in the substrate is equivalent to an efficiencyof fluorescence energy transfer of 96%. This large fluorescence changeupon β-lactam cleavage can readily be used to detect β-lactamase in thecytoplasm of living mammalian cells, as is reported in Examples 6 and 7.

The 7-hydroxycoumarin moiety in the cephalosporin was determined to havea fluorescence quantum efficiency in the absence of the acceptor of98-100%. This value was determined by comparing the integral of thecorrected fluorescence emission spectrum of the dye with that of asolution of 9-aminoacridine hydrochloride in water matched forabsorbance at the excitation wavelength. It follows that7-hydroxycoumarin is an ideal donor dye, as virtually every photonabsorbed by the dye undergoes fluorescence energy transfer to theacceptor.

Example 6

Cells of the T-cell lymphoma line Jurkat were suspended in an isotonicsaline solution (Hank's balanced salt solution) containing approximately10¹² β-lactamase enzyme molecules per milliliter (approximately 1.7 nM;Penicillinase 205 TEM R⁺, from Sigma) and 1 mg/ml rhodamine conjugatedto dextran (40 kd) as a marker of loading. The suspension was passedthrough a syringe needle (30 gauge) four times. This causes transient,survivable disruptions of the cells' plasma membrane and allows entry oflabeled dextran and β-lactamase. Cells that has been successfullypermeabilized contained β-lactamase and were red fluorescent whenilluminated at the rhodamine excitation wavelength on a fluorescentmicroscope. The cells were incubated with 5 μM fluorogenic β-lactamasesubstrate, CCF2/ac₂AM₂, at room temperature for 30 minutes. Illuminationwith violet light (405 nm) revealed blue fluorescent and greenfluorescent cells. All cells that had taken up the markerrhodamine-dextran appeared fluorescent blue, while cells devoid theenzyme appeared fluorescent green.

Example 7

Cells from cell lines of various mammalian origin were transientlytransfected with a plasmid containing the RTEM β-lactamase gene underthe control of a mammalian promotor. The gene encodes cytosolicβ-lactamase lacking any signal sequence and is listed as SEQ. ID. 1. 10to 48 hours after transfection cells were exposed to 5 μmol CCF2/ac₂AM₂for 1 to 6 hours. In all cases fluorescent blue cells were detected onexamination with a fluorescence microscope. Not a single bluefluorescent cell was ever detected in nontransfected control cells. Toquantitate the fluorescence measurements the cells were first viewedthrough coumarin (450 DF 65) and then fluorescein (515 EFLP) emissionfilters and pictures were recorded with a charge couple device camera.The average pixel intensities of CCF2 loaded transfected cells (blue)and controls (green) at coumarin and fluorescein wavelength in COS-7(Table 2) and CHO (Table 3) cells are summarized; values for 4representative cells for each population are given. Thus, the substrateCCF2 revealed gene expression in single living mammalian cells.

TABLE 2 COS-7 (origin: SV40 transformed african green monkey kidneycells) Table of pixel coumarin fluorescein intensities emission filteremission filter Blue cell #1 27 20 #2 34 23 #3 31 31 #4 22 33 Green cell#1 4 43 #2 4 42 #3 5 20 #4 3 24

TABLE 2 COS-7 (origin: SV40 transformed african green monkey kidneycells) Table of pixel coumarin fluorescein intensities emission filteremission filter Blue cell #1 27 20 #2 34 23 #3 31 31 #4 22 33 Green cell#1 4 43 #2 4 42 #3 5 20 #4 3 24

Example 8

For preparation of7-acetyloxy-3-(N-carboxymethyl-N-methylaminocarbonyl)coumarin, 400 mg(1.6 mMol) 3-carboxy-7-acetylcoumarin were refluxed with 4 ml thionylchloride for 20 minutes. Excess thionyl chloride was removed bydistillation and the residue (7-acetyloxy-3-chlorocarbonylcoumarin)stored in vacuo over potassium hydroxide pellets overnight. In aseparate vessel 142.5 mg (1.6 mMol) sarcosine was dissolved in 1.05 ml(5.4 mMol) N-methyl trimethylsilyl trifluorooacetamide (MSTFA) and keptat room temperature for 16 hours. 2 ml dry acetonitrile and 187 μl (1.7mMol) N-methylmorpholine were added and the solution was poured onto thesolid 7-acetyloxy-3-chlorocarbonylcoumarin on ice. After stirring for 20minutes on ice the solution was let to warm to room temperature. After 4hours the solvents were removed in vacuo. The residue was dissolved inmethanol to deprotect the acid after which the solvent was removed invacuo. The solid was dissolved in 30 ml ethylacetate-acetonitrile (2:1)and the solution extracted twice with an equal volume of 1 Nhydrochloric acid and the with brine. The organic phase was dried overanhydrous sodium sulfate. The solvent was removed in vacuo and the solidcrystallized from boiling ethylacetate with addition of hexane. Theyield was 316 mg (1.0 mMol, 63%) of a white crystalline solid.

Coupling of7-acetyloxy-3-(N-carboxymethyl-N-methylaminocarbonyl)coumarin with7-amino-3′-chlorocephalosporanic acid benzhydryl ester was effected asfollows. 62 mg (0.2 mMol)7-acetyloxy-3-(N-carboxymethyl-N-methylaminocarbonyl)coumarin wasstirred with 1 ml dry methylene chloride to which 27 mg (0.2 mMol)hydroxybenztriazole and 41 mg dicyclohexyl carbodiimide had been added.A solution of 82.6 mg (0.2 mMol) 7-amino 3′-chloro cephalosporanic acidbenzhydryl ester in 1 ml methylene chloride was added dropwise over aperiod of 5 minutes. The reaction was stirred for 20 hours at roomtemperature after which the precipitate was removed by filtration. Thefiltrate was evaporated in vacuo and the product extracted intomethylene chloride. The solvent was removed once more and the residuedissolved in 1 ml ethyl acetate. Addition of three volumes of hexaneprecipitated the product which was recovered by centrifugation. Theyield was 49.9 mg (70 μMol, 35%) of the product as a white powder.

Conversion of the cephalosporin 3′-chloro substituent in the aboveproduct to the 3′-iodo substituent was carried out as follows. 49.9 mg(70 μMol) of the above product was stirred with 52.5 mg sodium iodide (5equivalents) in 1.2 ml dry methyl ethyl ketone at room temperature for 2hours. The solvent was removed in vacuo and the residue dissolved in 2ml ethyl acetate—methylene chloride (1:1) and extracted with cold 2%aqueous sodium thiosulfate solution, followed by two extractions withbrine. The organic layer was dried over anhydrous sodium sulfate. Theslightly orange powder (32 mg, 40 μMol, 57%) was used without furtherpurification in the next reaction.

Coupling of above product with 5-merceptofluorescein diacetate (productCCF1ac₃ diphenylmethyl ester) was effected by dissolving 32 mg (40 μMol)of the iodo derivative in 0.4 ml dimethylformamide and 3.4 mg sodiumbicarbonate added. 22 mg (50 μMol) 5-mercaptofluorescein diacetate weredissolved in 0.3 ml deoxygenated dimethyl formamide and added to theiodo compound in an argon atmosphere. After 2 hours the solvent wasremoved in vacuo. The residue was suspended in methylene chloride—ethylacetate (1:1). The organic solution was washed with water and dried overanhydrous sodium sulfate. The solvent was removed and the residue wastriturated with ethyl ether hexane (1:1). Flash chromatography on 60mesh silica gel with ethyl acetate—toluene (2:1) yielded 4.2 mg (4 μMol,10%) of colorless product.

Cleavage of the diphenylmethyl ester to give CCF1ac₃ was effected asfollows. 4 mg (4 μMol) of CCF1ac₃ diphenylmethyl ester were treated with200 μl trifluoroacetic acid—anisole—methylene chloride (10:1:10) on icefor 15 minutes. The reagents were removed in vacuo and the residue wasdissolved in 0.5 ml ethyl acetate and the solvent evaporated in vacuo.The solid was triturated with ether and then dissolved in 0.5 mlmethanol. Addition of the methanolic solution to 2 ml water precipitatedthe product. The product was recovered by centrifugation and dried invacuo. The yield was 2 mg (2 μMol. 50%) white solid. The compound wasfurther purified by high performance liquid chromatography on a reversephase C₁₈-column using 55% aqueous acetonitrile containing 0.5% aceticacid as the eluent.

The fluorescence emission spectrum of CCF1 before and after β-lactamasecleavage (FIG. 6) was obtained from a sample of CCF1ac₃ that had beenconverted to CCF1 by treatment with orange peel acetyl esterase in 50mmolar aqueous phosphate buffer pH 7.

Substrate CCF1 has similar fluorescence properties to substrate CCF2 inExample 5. In the intact substrate, efficient energy transfer occursfrom the 7-hydroxycoumarin moiety to the fluorescein moiety. Excitationof the substrate at 390 nm results in fluorescence emission at 515 nm(green) from the acceptor dye fluorescein. The energy transfer isdisrupted when β-lactamase cleaves the β-lactam ring, thereby severingthe link between the two dyes. Excitation of the products at 390 nm nowresults entirely in donor fluorescence emission at 460 nm (blue). Thefluorescence emission from the donor moiety increases 25-fold uponβ-lactam cleavage. The fluorescence at 515 nm is reduced by 3-fold, allof the remaining fluorescence originating from the 7-hydroxycoumarin asits emission spectrum extends into the green. Twenty-five-fold quenchingof the donor in the substrate is equivalent to an efficiency offluorescence energy transfer of 96%. This large fluorescence change uponβ-lactam cleavage can readily be used to detect β-lactamase in thecytoplasm of living mammalian cells, as is reported in Example 9.

Example 9

Cells of the T-cell lymphoma line Jurkat were suspended in an isotonicsaline solution (hank's balanced salt solution) containing approximately10¹² β-lactamase enzyme molecules per milliliter (approximately 1.7 nM;Penicillinase 205 TEM R⁺, from Sigma) and 1 mg/ml rhodamine conjugatedto dextran (40 kd) as a marker of loading. The suspension was passedthrough a syringe needle (30 gauge) four times. This causes transient,survivable disruptions of the cells' plasma membrane and allows entry oflabeled dextran and β-lactamase. Cells which had been successfullypermeabilized contained β-lactamase and were red fluorescent whenilluminated at the rhodamine excitation wavelength on a fluorescentmicroscope. The cells were incubated with 30 μM fluorogenic β-lactamasesubstrate CCF1ac₃ at room temperature for 30 minutes. Illumination withultraviolet light (360 nm) revealed blue fluorescent and greenfluorescent cells. All cells that had taken up the markerrhodamine-dextran appeared fluorescent blue, while cells devoid theenzyme appeared fluorescent green.

Example 10

The preferred membrane-permeable ester of CCF2 was prepared as follows:

Coupling of 5-fluoroesceinthiol diacetate (5) and7-amino-3′-chlorocephalosporanic acid benzhydryl ester was effected asfollows. 450 mg (1 mmol) 5-mercaptofluorescein diacetate disulfide dimerwere dissolved in 30 ml chloroform and treated with 50 μl water and 125μl tributylphosphine in a nitrogen atmosphere which generated the free5-fluoresceinthiol. 450 mg (1 mmol) 7-Amino-3′-chlorocephalosporanicacid benzhydryl ester hydrochloride salt were dissolved in 10 mlacetonitrile with the help of 220 μl (2 mmol) N-methyl morpholine andthe two solutions combined after 30 minutes. One hour later the solventvolume was reduced to 5 ml and 50 ml carbon tetrachloride were added.The solvent volume was reduced to 15 ml and hexane added with stirring.The initial orange precipitate consisting mainly of N-methyl morpholinehydrochloride was removed by filtration. Upon further addition of twovolumes of hexane 630 mg (0.76 mmol, 76%) white product was precipitatedand collected.

The above product was coupled with 7-butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin. 325 mg (0.88 mmol)7-Butyryloxy-3-carboxymethyl aminocarbonyl-6-chlorocoumarin wasdissolved in 15 ml hot dry dioxane. With rapid cooling 110 μl (1 mmol)N-methyl morpholine in 1 ml dioxane and 115 μl (0.9 mmol) isobutylchloroformate in 8 ml methylene chloride were added. The reaction waskept at 0° C. for 30 minutes after which 661 mg (0.8 mmol) of the abovefluorescein-cephalosporin adduct in 7 ml dry methylene chloride wereadded. The solution was let to warm to room temperature and after 3hours the solvents were removed in vaccuo. The residue was dissolved in30 ml methylene chloride and twice extracted with one volume 10% aqueousacetic acid and once with water. The organic phase was dried overanhydrous sodium sulfate. Addition of 150 ml dry ethanol, reduction ofthe solvent volume to 50 ml and cooling to −20° C. resulted inprecipitation of the product (crude, 850 mg). Purification was achievedby chromatography over silica gel with 25% ethyl acetate in toluene asthe eluent. 250 mg (0.21 mmol, 26%) of white powderous product wascollected.

Cleavage of the cephalosporin benzhydryl ester was accomplished bytreatment with trifluoroacetic acid. 145 mg (0.12 mmol) of the aboveproduct was treated with trifluoroacetic acid/methylene chloride/anisole(10/10/1) at 0° C. for 20 minutes. The reagents were removed in vacuoand the residue triturated with diisopropyl ether. The solid wasdissolved in 1 ml dimethyl sulfoxide and the product precipitated byaddition to 25 ml water. It was further purified on reverse phase C₁₈resin with a step gradient of 40 to 60% aqueous acetonitrile containing0.5% acetic acid as the eluent yielding 74 mg (73 μmol, 60%) whitepowder.

Protection of cephalosporin acid as membrane permeable acetoxymethylester was achieved as follows. 15 mg (15 μmol) of the above product wasdissolved in 250 μl methylene chloride. 25 μl Bromomethyl acetate and 50μl lutidine were added to the solution. The reaction was kept at ambienttemperature for 7 hours after which the reagents were removed in vacuo.The residue was purified by flash chromatography on silica gel withethyl acetate as the eluent. 15 mg (14 μmol, 92%) white product wasobtained. This compound, named CCF2/btAMac₂ was used for intracellulardetection of β-lactamase activity.

Example 11

Measurement of activation of an intracellular receptor: Activation ofthe intracellular glucocorticoid receptor was measured by its ability toupregulate the transcriptional activity of the glucocorticoid responsiveelement in the mouse mammary tumor virus promotor. This response tosteroids was detected as increased intracellular β-lactamase activity onthe substrate CCF2 causing an appropriate change in fluorescent signal.

The gene for plasmid encoded RTEM β-lactamase of Escherichia coliwithout a signal sequence (Sequence 1 of FIG. 7) was put undertranscriptional control of the mouse mammary tumor virus promotor andintroduced into a mammalian expression vector. This vector also carriedthe chloroamphenicol resistance marker for amplification of the plasmidin bacteria and the neomycin resistance marker for mammalian selection.It was introduced into baby hamster kidney (BHK) cells in culture usingthe calcium phosphate precipitation technique. Cells were then subjectedto selection for stable integration of the plasmid into the cells'genome using the antibiotic G418. One of twenty clones was selected forits marked increase in β-lactamase expression following exposure to thesteroid analog dexamethasone.

The following describes the measurement of the increase in β-lactamasegene expression in this clone after addition of the agonistdexamethasone. Cells of the stable BHK cell clone G941 expressingβ-lactamase under control of the glucocorticoid-inducible promotor werekept in the presence or absence of the agonists in the incubator at 37°C. Flasks with cells were removed from the incubator at differentintervals after agonist addition and the cells transferred into Hank'sbalanced salt solution containing 10 μmolar CCF2/btAMac₂. This compoundbecomes converted to the β-lactamase accessible fluorescent substrateCCF2 by endogenous cytoplasmic esterases. Ten minutes later the cellsupernatant containing CCF2/btAMac₂ was removed. 30 Minutes later thecells were imaged with a cooled CCD camera mounted on an epifluorescencemicroscope. Fluorescence measurements were taken with violet excitationlight (filter 400DF15) and with blue (filter 450DF65) and green (filter535DF45) emission filters. A ratio of blue versus green emissionintensities was determined. The ratio is a measure of how much substratehas been converted to product. Using a 40× objective, 4 fields withapproximately 60 cells each were imaged at each time point. The resultsshow a significant increase in the ratio of fluorescent intensitiesreflective of increasing β-lactamase expression and production.

time in presence of 1 μM dexamethasone 0.0 hours 1.0 hours 2.0 hours 3.3hours average ratio 0.21 +/− 0.38 +/− 0.42 +/− 0.47 +/− of fluorescence0.02 0.05 0.07 0.08 intensities 450DF65/ 535DF45

Example 12

Measurement of cell surface receptor activation and intracellularsignaling via second-messenger responsive elements: Activation of cellsurface receptors leads to a change in intracellular messengerconcentrations which in turn modulates intracellular transcriptionfactor activity. In lymphocytes, an increase the intracellularconcentration of the messenger ion calcium leads to the activation ofthe nuclear factor of activated T-lymphocytes (NFAT). This eventincreases transcription at promoters containing the NFAT-recognitionsite. An increase in calcium levels alone is sufficient to markedlyincrease transcription of a reporter gene such as β-lactamase regulatedwhen it is put under transcriptional control of a promotor containing atrimer of NFAT sites.

The murine T-lymphocyte cell line B3Z was transiently cotransfected withtwo plasmids. One plasmid contained the β-adrenergic receptor, whichlocalizes at the cells' surface, under the transcriptional control ofthe strong and constitutively active cytomegalovirus (CMV) promoter. Theother plasmid contained the bacterial RTEM β-lactamase gene fromEscherichia coli modified for improved mammalian expression (sequence ID#3, with optimum mammalian Kozak sequence, β-globin leader sequence,pre-sequence removed) under the transcriptional control of a promotorcontaining a trimer of NFAT sites. The plasmids were introduced intocells using electroporation. 5×10⁶ cells in 0.5 ml electroporationbuffer were electroporated in the presence of 10 μg each of bothplasmids using the Biorad Gene Pulser (250V, 960 μF, 16 μsec).Twenty-four hours after transfection, cells were either incubated in thepresence or absence of the β-adrenergic agonist isoproterenol (10μmolar) for 5 hours. The supernatant was removed and replaced withHank's balanced salt solution containing 10 μmolar CCF2/btAMac₂. After20 minutes at room temperature cells were washed with fresh buffer andviewed with the fluorescence microscope. 4% of isoproterenol treatedcells appeared fluorescent blue (excitation filter 400DF15, emissionfilter 435 nm longpass) while no blue fluorescent cells were detectablein the control population (absence of agonist). Maximal stimulation with2 μM ionomycin and 50 ng/ml phorbol ester for 5 hours resulted in 20%blue fluorescent cells in the population.

Example 13

β-Lactamases from different microorganisms were modified for use asreporter enzymes in eukaryotic cells, preferably mammalian. Thebacterial gene for these enzymes includes a N-terminal pre-sequence(first 23 amino acids of Sequence 2 of FIG. 7.) that targets the enzymeto the extracellular space. Following translocation a pre-sequencepeptidase cleaves the 23 amino acid pre-sequence releasing the matureβ-lactamase enzyme. RTEM β-lactamase from Escherichia coli including itsbacterial pre-sequence (Sequence 2 of FIG. 7) was put into a mammalianexpression vector under the control of the mouse mammary tumor viruspromotor. This construct was introduced into baby hamster kidney cellsusing the standard calcium phosphate precipitation technique. Theβ-lactamase activity was found in the cell culture medium; no activitycould be detected in the cell pellet. The amount of β-lactamase activityin the medium was steroid dependent. Cells that had been in the presenceof 1 μM dexamethasone for 36 hours prior to the measurement producedthreefold more enzyme than control. This makes the β-lactamase with itsbacterial pre-sequence (Sequences 2 of FIG. 7) useful for anextracellular assay of mammalian reporter gene activity.

A preferred use of the β-lactamase reporter is where the enzyme isproduced and retained in the cell cytoplasm. Therefore the bacterialsignal sequence was removed and replaced by ATG (methionine) as the newtranslational start site in three modified RTEM β-lactamase genes(Sequences 1, 3, and 4 of FIG. 7). In order to increase expression ofthe β-lactamases in mammalian cells, the RTEM β-lactamases of Sequence 3and 4 of FIG. 7 were constructed with altered ribosome binding sitesoptimized for mammalian expression [Kozak, M., J. Cell Biol. 108:229-241 (1989)]. For increased compatibility with the mammaliantranslation machinery, β-lactamase of sequence ID #3 was inserted at theend of an untranslated mammalian β-globin leader sequence. All of thesenovel DNA sequences encoding novel β-lactamases were inserted intomammalian expression vectors with the cytomegalovirus promotorcontrolling their transcription. Mammalian cells in tissue culture(Hela, COS-7, CHO, BHK) were transfected transiently with the plasmidsusing the standard lipofectin technique. Two to five days aftertransfection, the cells were incubated with the membrane-permeantderivative, CCF2/btAMac₂, of the fluorescent substrate CCF2 to assayfunctional expression of the enzyme. 5-20% of cells transfected withplasmids containing cDNA Sequences 2, 3 and 4 of FIG. 7 showed aconversion of green to blue fluorescence indicating cleavage of theintracellularly trapped substrate by expressed β-lactamase. By contrast,in untransfected or mock transfected controls, all cells showed thegreen fluorescence of uncleaved CCF2; no blue-fluorescing cells wereobserved, confirming the absence of any endogenous β-lactamase activity.

The gene for Bacillus licheniformis β-lactamase was isolated from totalBacillus licheniformis DNA by use of the polymerase chain reaction. Theoligonucloeotide primers removed the β-lactamase secretion sequence andgenerated the DNA sequence ID #5. This gene was inserted in a pCDNA3mammalian expression vector under the transcriptional control of theconstitutively active cytomegalovirus promoter. HeLa cells weretransfected with 10 μg of plasmid per 25 cm² culture dish usinglipofectin. 5 days after transfection, cells were tested for functionalexpression of β-lactamase by incubating them in the presence of 100μmolar CCF2/btAMac₂ and visual inspection with the epifluorescencemicroscope. 30-40% of cells showed blue fluorescence, whereas onlygreen-fluorescing cells, no blue-fluorescing cells were detectable inuntransfected controls. In transient transfections, it is typical for<50% of the cells to become transfected.

Example 14

A plasmid was constructed with β-lactamase of sequence ID 3 (FIG. 7)under control of yeast elongation factor EF-1alpha enhancer andpromoter. This plasmid was coinjected together with the potassium saltof substrate CCF2 (compound 7b) into zebrafish embryos at the singlecell stage. As control, embryos were injected with the potassium salt ofsubstrate CCF2 alone. After three hours, the embryos were viewed with anepifluorescence microscope using violet excitation light (filter400DF15) and a 435 nm longpass emission filter. Embryos that hadreceived plasmid DNA fluoresced blue while controls fluoresced green.

Example 15

The β-lactamase gene of sequence ID 3 was cloned into a Drosophilatransformation vector under the control of the glass promotor andinjected into wild-type Drosophila embryos. As control, the β-lactamasegene was inserted in the wrong orientation. Drosophila embryos weregermline-transformed using P element-mediated transformation. Thetransformations and all subsequent fly manipulations were performedusing standard techniques [Karess, R. E. and Rubin, G. M., Cell 38, 135,(1984)]. Omatidia of late stage transformed pupe were transsected anddissociated to single cells. The cells were incubated in buffer with 40μmolar CCF2/btAMac₂ (compound 34) for 20 minutes, washed and viewed withan epifluorescence microscope (excitation filter 400DF15, emissionfilter 435 nm long pass). Omatidia cells from flyes transformed with theβ-lactamase gene in the proper orientation fluoresced blue, whileomatidia cells containing the gene in the wrong orientation fluorescedgreen.

Example 16

In certain embodiments the compound of this invention can be any of thefollowing compounds.

wherein

R^(Y) is selected from the group consisting of H, Cl, and Br;

R^(X) is selected from the group consisting of H, and methyl;

wherein R^(z) and R^(z1) are independently selected from the groupconsisting of —C(O)alk, —CH₂OC(O)alk, —CH₂SC(O)alk, —CH₂OC(O))alk, loweracyloxy-alpha-benzyl, and deltrabutyrolactonyl; wherein alk is loweralkyl of 1 to 4 carbon atoms, and membrane-permeant fluorogenicderivatives thereof,

R″ is 1-(acyloxy)alkyl.

Another example of the compound is:

wherein R^(z) and R^(z1) are independently selected from the groupconsisting of —C(O)alk, —CH₂OC(O)alk, —CH₂SC(O)alk, —CH₂OC(O))alk, loweracyloxy-alpha-benzyl, and deltabutyrolactonyl; wherein alk is loweralkyl of 1 to 4 carbon atoms.

Another example of the compound is:

A final example of the compound is:

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

The present invention provides novel substrates for beta-lactamase,beta-lactamases and methods for their use. While specific examples havebeen provided, the above description is illustrative and notrestrictive. Many variations of the invention will become apparent tothose skilled in the art upon review of this specification. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

10 795 base pairs nucleic acid double linear Genomic DNA not providedCoding Sequence 1...795 1 ATG AGT CAC CCA GAA ACG CTG GTG AAA GTA AAAGAT GCT GAA GAT CAG 48 Met Ser His Pro Glu Thr Leu Val Lys Val Lys AspAla Glu Asp Gln 1 5 10 15 TTG GGT GCA CGA GTG GGT TAC ATC GAA CTG GATCTC AAC AGC GGT AAG 96 Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp LeuAsn Ser Gly Lys 20 25 30 ATC CTT GAG AGT TTT CGC CCC GAA GAA CGT TTT CCAATG ATG AGC ACT 144 Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro MetMet Ser Thr 35 40 45 TTT AAA GTT CTG CTA TGT GGC GCG GTA TTA TCC CGT GTTGAC GCC GGG 192 Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Val AspAla Gly 50 55 60 CAA GAG CAA CTC GGT CGC CGC ATA CAC TAT TCT CAG AAT GACTTG GTT 240 Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp LeuVal 65 70 75 80 GAG TAC TCA CCA GTC ACA GAA AAG CAT CTT ACG GAT GGC ATGACA GTA 288 Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met ThrVal 85 90 95 AGA GAA TTA TGC AGT GCT GCC ATA ACC ATG AGT GAT AAC ACT GCGGCC 336 Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala100 105 110 AAC TTA CTT CTG ACA ACG ATC GGA GGA CCG AAG GAG CTA ACC GCTTTT 384 Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe115 120 125 TTG CAC AAC ATG GGG GAT CAT GTA ACT CGC CTT GAT CGT TGG GAACCG 432 Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro130 135 140 GAG CTG AAT GAA GCC ATA CCA AAC GAC GAG CGT GAC ACC ACG ATGCCT 480 Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro145 150 155 160 GCA GCA ATG GCA ACA ACG TTG CGC AAA CTA TTA ACT GGC GAACTA CTT 528 Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu LeuLeu 165 170 175 ACT CTA GCT TCC CGG CAA CAA TTA ATA GAC TGG ATG GAG GCGGAT AAA 576 Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala AspLys 180 185 190 GTT GCA GGA CCA CTT CTG CGC TCG GCC CTT CCG GCT GGC TGGTTT ATT 624 Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp PheIle 195 200 205 GCT GAT AAA TCT GGA GCC GGT GAG CGT GGG TCT CGC GGT ATCATT GCA 672 Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile IleAla 210 215 220 GCA CTG GGG CCA GAT GGT AAG CCC TCC CGT ATC GTA GTT ATCTAC ACG 720 Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile TyrThr 225 230 235 240 ACG GGG AGT CAG GCA ACT ATG GAT GAA CGA AAT AGA CAGATC GCT GAG 768 Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln IleAla Glu 245 250 255 ATA GGT GCC TCA CTG ATT AAG CAT TGG 795 Ile Gly AlaSer Leu Ile Lys His Trp 260 265 265 amino acids amino acid linearprotein internal not provided 2 Met Ser His Pro Glu Thr Leu Val Lys ValLys Asp Ala Glu Asp Gln 1 5 10 15 Leu Gly Ala Arg Val Gly Tyr Ile GluLeu Asp Leu Asn Ser Gly Lys 20 25 30 Ile Leu Glu Ser Phe Arg Pro Glu GluArg Phe Pro Met Met Ser Thr 35 40 45 Phe Lys Val Leu Leu Cys Gly Ala ValLeu Ser Arg Val Asp Ala Gly 50 55 60 Gln Glu Gln Leu Gly Arg Arg Ile HisTyr Ser Gln Asn Asp Leu Val 65 70 75 80 Glu Tyr Ser Pro Val Thr Glu LysHis Leu Thr Asp Gly Met Thr Val 85 90 95 Arg Glu Leu Cys Ser Ala Ala IleThr Met Ser Asp Asn Thr Ala Ala 100 105 110 Asn Leu Leu Leu Thr Thr IleGly Gly Pro Lys Glu Leu Thr Ala Phe 115 120 125 Leu His Asn Met Gly AspHis Val Thr Arg Leu Asp Arg Trp Glu Pro 130 135 140 Glu Leu Asn Glu AlaIle Pro Asn Asp Glu Arg Asp Thr Thr Met Pro 145 150 155 160 Ala Ala MetAla Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu 165 170 175 Thr LeuAla Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys 180 185 190 ValAla Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile 195 200 205Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala 210 215220 Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr 225230 235 240 Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile AlaGlu 245 250 255 Ile Gly Ala Ser Leu Ile Lys His Trp 260 265 858 basepairs nucleic acid double linear Genomic DNA not provided CodingSequence 1...858 3 ATG AGA ATT CAA CAT TTC CGT GTC GCC CTT ATT CCC TTTTTT GCG GCA 48 Met Arg Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe PheAla Ala 1 5 10 15 TTT TGC CTT CCT GTT TTT GGT CAC CCA GAA ACG CTG GTGAAA GTA AAA 96 Phe Cys Leu Pro Val Phe Gly His Pro Glu Thr Leu Val LysVal Lys 20 25 30 GAT GCT GAA GAT CAG TTG GGT GCA CGA GTG GGT TAC ATC GAACTG GAT 144 Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu LeuAsp 35 40 45 CTC AAC AGC GGT AAG ATC CTT GAG AGT TTT CGC CCC GAA GAA CGTTTT 192 Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe50 55 60 CCA ATG ATG AGC ACT TTT AAA GTT CTG CTA TGT GGC GCG GTA TTA TCC240 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 6570 75 80 CGT GTT GAC GCC GGG CAA GAG CAA CTC GGT CGC CGC ATA CAC TAT TCT288 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 8590 95 CAG AAT GAC TTG GTT GAG TAC TCA CCA GTC ACA GAA AAG CAT CTT ACG336 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100105 110 GAT GGC ATG ACA GTA AGA GAA TTA TGC AGT GCT GCC ATA ACC ATG AGT384 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115120 125 GAT AAC ACT GCG GCC AAC TTA CTT CTG ACA ACG ATC GGA GGA CCG AAG432 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130135 140 GAG CTA ACC GCT TTT TTG CAC AAC ATG GGG GAT CAT GTA ACT CGC CTT480 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 145150 155 160 GAT CGT TGG GAA CCG GAG CTG AAT GAA GCC ATA CCA AAC GAC GAGCGT 528 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg165 170 175 GAC ACC ACG ATG CCT GCA GCA ATG GCA ACA ACG TTG CGC AAA CTATTA 576 Asp Thr Thr Met Pro Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu180 185 190 ACT GGC GAA CTA CTT ACT CTA GCT TCC CGG CAA CAA TTA ATA GACTGG 624 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp195 200 205 ATG GAG GCG GAT AAA GTT GCA GGA CCA CTT CTG CGC TCG GCC CTTCCG 672 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro210 215 220 GCT GGC TGG TTT ATT GCT GAT AAA TCT GGA GCC GGT GAG CGT GGGTCT 720 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser225 230 235 240 CGC GGT ATC ATT GCA GCA CTG GGG CCA GAT GGT AAG CCC TCCCGT ATC 768 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser ArgIle 245 250 255 GTA GTT ATC TAC ACG ACG GGG AGT CAG GCA ACT ATG GAT GAACGA AAT 816 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu ArgAsn 260 265 270 AGA CAG ATC GCT GAG ATA GGT GCC TCA CTG ATT AAG CAT TGG858 Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285286 amino acids amino acid linear protein internal not provided 4 MetArg Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15Phe Cys Leu Pro Val Phe Gly His Pro Glu Thr Leu Val Lys Val Lys 20 25 30Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 7580 Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 9095 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly ProLys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val ThrArg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile ProAsn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro Ala Ala Met Ala Thr ThrLeu Arg Lys Leu Leu 180 185 190 Thr Gly Glu Leu Leu Thr Leu Ala Ser ArgGln Gln Leu Ile Asp Trp 195 200 205 Met Glu Ala Asp Lys Val Ala Gly ProLeu Leu Arg Ser Ala Leu Pro 210 215 220 Ala Gly Trp Phe Ile Ala Asp LysSer Gly Ala Gly Glu Arg Gly Ser 225 230 235 240 Arg Gly Ile Ile Ala AlaLeu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255 Val Val Ile Tyr ThrThr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270 Arg Gln Ile AlaGlu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 285 843 base pairsnucleic acid double linear Genomic DNA not provided Coding Sequence49...843 5 AAGCTTTTTG CAGAAGCTCA GAATAAACGC AACTTTCCGG GTACCACC ATG GGGCAC 57 Met Gly His 1 CCA GAA ACG CTG GTG AAA GTA AAA GAT GCT GAA GAT CAGTTG GGT GCA 105 Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln LeuGly Ala 5 10 15 CGA GTG GGT TAC ATC GAA CTG GAT CTC AAC AGC GGT AAG ATCCTT GAG 153 Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile LeuGlu 20 25 30 35 AGT TTT CGC CCC GAA GAA CGT TTT CCA ATG ATG AGC ACT TTTAAA GTT 201 Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe LysVal 40 45 50 CTG CTA TGT GGC GCG GTA TTA TCC CGT GAT GAC GCC GGG CAA GAGCAA 249 Leu Leu Cys Gly Ala Val Leu Ser Arg Asp Asp Ala Gly Gln Glu Gln55 60 65 CTC GGT CGC CGC ATA CAC TAT TCT CAG AAT GAC TTG GTT GAG TAC TCA297 Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser 7075 80 CCA GTC ACA GAA AAG CAT CTT ACG GAT GGC ATG ACA GTA AGA GAA TTA345 Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu 8590 95 TGC AGT GCT GCC ATA ACC ATG AGT GAT AAC ACT GCG GCC AAC TTA CTT393 Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu 100105 110 115 CTG ACA ACG ATC GGA GGA CCG AAG GAG CTA ACC GCT TTT TTG CACAAC 441 Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn120 125 130 ATG GGG GAT CAT GTA ACT CGC CTT GAT CAT TGG GAA CCG GAG CTGAAT 489 Met Gly Asp His Val Thr Arg Leu Asp His Trp Glu Pro Glu Leu Asn135 140 145 GAA GCC ATA CCA AAC GAC GAG CGT GAC ACC ACG ATG CCT GTA GCAATG 537 Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met150 155 160 GCA ACA ACG TTG CGC AAA CTA TTA ACT GGC GAA CTA CTT ACT CTAGCT 585 Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala165 170 175 TCC CGG CAA CAA TTA ATA GAC TGG ATG GAG GCG GAT AAA GTT GCAGGA 633 Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly180 185 190 195 CCA CTT CTG CGC TCG GCC CTT CCG GCT GGC TGG TTT ATT GCTGAT AAA 681 Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala AspLys 200 205 210 TCT GGA GCC GGT GAG CGT GGG TCT CGC GGT ATC ATT GCA GCACTG GGG 729 Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala LeuGly 215 220 225 CCA GAT GGT AAG CCC TCC CGT ATC GTA GTT ATC TAC ACG ACGGGG AGT 777 Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr GlySer 230 235 240 CAG GCA ACT ATG GAT GAA CGA AAT AGA CAG ATC GCT GAG ATAGGT GCC 825 Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile GlyAla 245 250 255 TCA CTG ATT AAG CAT TGG 843 Ser Leu Ile Lys His Trp 260265 265 amino acids amino acid linear protein internal not provided 6Met Gly His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln 1 5 1015 Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys 20 2530 Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr 35 4045 Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Asp Asp Ala Gly 50 5560 Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val 65 7075 80 Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val 8590 95 Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala100 105 110 Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr AlaPhe 115 120 125 Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp His TrpGlu Pro 130 135 140 Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp ThrThr Met Pro 145 150 155 160 Val Ala Met Ala Thr Thr Leu Arg Lys Leu LeuThr Gly Glu Leu Leu 165 170 175 Thr Leu Ala Ser Arg Gln Gln Leu Ile AspTrp Met Glu Ala Asp Lys 180 185 190 Val Ala Gly Pro Leu Leu Arg Ser AlaLeu Pro Ala Gly Trp Phe Ile 195 200 205 Ala Asp Lys Ser Gly Ala Gly GluArg Gly Ser Arg Gly Ile Ile Ala 210 215 220 Ala Leu Gly Pro Asp Gly LysPro Ser Arg Ile Val Val Ile Tyr Thr 225 230 235 240 Thr Gly Ser Gln AlaThr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu 245 250 255 Ile Gly Ala SerLeu Ile Lys His Trp 260 265 792 base pairs nucleic acid double linearGenomic DNA not provided Coding Sequence 1...792 7 ATG GAC CCA GAA ACGCTG GTG AAA GTA AAA GAT GCT GAA GAT CAG TTG 48 Met Asp Pro Glu Thr LeuVal Lys Val Lys Asp Ala Glu Asp Gln Leu 1 5 10 15 GGT GCA CGA GTG GGTTAC ATC GAA CTG GAT CTC AAC AGC GGT AAG ATC 96 Gly Ala Arg Val Gly TyrIle Glu Leu Asp Leu Asn Ser Gly Lys Ile 20 25 30 CTT GAG AGT TTT CGC CCCGAA GAA CGT TTT CCA ATG ATG AGC ACT TTT 144 Leu Glu Ser Phe Arg Pro GluGlu Arg Phe Pro Met Met Ser Thr Phe 35 40 45 AAA GTT CTG CTA TGT GGC GCGGTA TTA TCC CGT ATT GAC GCC GGG CAA 192 Lys Val Leu Leu Cys Gly Ala ValLeu Ser Arg Ile Asp Ala Gly Gln 50 55 60 GAG CAA CTC GGT CGC CGC ATA CACTAT TCT CAG AAT GAC TTG GTT GAG 240 Glu Gln Leu Gly Arg Arg Ile His TyrSer Gln Asn Asp Leu Val Glu 65 70 75 80 TAC TCA CCA GTC ACA GAA AAG CATCTT ACG GAT GGC ATG ACA GTA AGA 288 Tyr Ser Pro Val Thr Glu Lys His LeuThr Asp Gly Met Thr Val Arg 85 90 95 GAA TTA TGC AGT GCT GCC ATA ACC ATGAGT GAT AAC ACT GCG GCC AAC 336 Glu Leu Cys Ser Ala Ala Ile Thr Met SerAsp Asn Thr Ala Ala Asn 100 105 110 TTA CTT CTG ACA ACG ATC GGA GGA CCGAAG GAG CTA ACC GCT TTT TTG 384 Leu Leu Leu Thr Thr Ile Gly Gly Pro LysGlu Leu Thr Ala Phe Leu 115 120 125 CAC AAC ATG GGG GAT CAT GTA ACT CGCCTT GAT CAT TGG GAA CCG GAG 432 His Asn Met Gly Asp His Val Thr Arg LeuAsp His Trp Glu Pro Glu 130 135 140 CTG AAT GAA GCC ATA CCA AAC GAC GAGCGT GAC ACC ACG ATG CCT GTA 480 Leu Asn Glu Ala Ile Pro Asn Asp Glu ArgAsp Thr Thr Met Pro Val 145 150 155 160 GCA ATG GCA ACA ACG TTG CGC AAACTA TTA ACT GGC GAA CTA CTT ACT 528 Ala Met Ala Thr Thr Leu Arg Lys LeuLeu Thr Gly Glu Leu Leu Thr 165 170 175 CTA GCT TCC CGG CAA CAA TTA ATAGAC TGG ATG GAG GCG GAT AAA GTT 576 Leu Ala Ser Arg Gln Gln Leu Ile AspTrp Met Glu Ala Asp Lys Val 180 185 190 GCA GGA CCA CTT CTG CGC TCG GCCCTT CCG GCT GGC TGG TTT ATT GCT 624 Ala Gly Pro Leu Leu Arg Ser Ala LeuPro Ala Gly Trp Phe Ile Ala 195 200 205 GAT AAA TCT GGA GCC GGT GAG CGTGGG TCT CGC GGT ATC ATT GCA GCA 672 Asp Lys Ser Gly Ala Gly Glu Arg GlySer Arg Gly Ile Ile Ala Ala 210 215 220 CTG GGG CCA GAT GGT AAG CCC TCCCGT ATC GTA GTT ATC TAC ACG ACG 720 Leu Gly Pro Asp Gly Lys Pro Ser ArgIle Val Val Ile Tyr Thr Thr 225 230 235 240 GGG AGT CAG GCA ACT ATG GATGAA CGA AAT AGA CAG ATC GCT GAG ATA 768 Gly Ser Gln Ala Thr Met Asp GluArg Asn Arg Gln Ile Ala Glu Ile 245 250 255 GGT GCC TCA CTG ATT AAG CATTGG 792 Gly Ala Ser Leu Ile Lys His Trp 260 264 amino acids amino acidlinear protein internal not provided 8 Met Asp Pro Glu Thr Leu Val LysVal Lys Asp Ala Glu Asp Gln Leu 1 5 10 15 Gly Ala Arg Val Gly Tyr IleGlu Leu Asp Leu Asn Ser Gly Lys Ile 20 25 30 Leu Glu Ser Phe Arg Pro GluGlu Arg Phe Pro Met Met Ser Thr Phe 35 40 45 Lys Val Leu Leu Cys Gly AlaVal Leu Ser Arg Ile Asp Ala Gly Gln 50 55 60 Glu Gln Leu Gly Arg Arg IleHis Tyr Ser Gln Asn Asp Leu Val Glu 65 70 75 80 Tyr Ser Pro Val Thr GluLys His Leu Thr Asp Gly Met Thr Val Arg 85 90 95 Glu Leu Cys Ser Ala AlaIle Thr Met Ser Asp Asn Thr Ala Ala Asn 100 105 110 Leu Leu Leu Thr ThrIle Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu 115 120 125 His Asn Met GlyAsp His Val Thr Arg Leu Asp His Trp Glu Pro Glu 130 135 140 Leu Asn GluAla Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val 145 150 155 160 AlaMet Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr 165 170 175Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val 180 185190 Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala 195200 205 Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala210 215 220 Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr ThrThr 225 230 235 240 Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln IleAla Glu Ile 245 250 255 Gly Ala Ser Leu Ile Lys His Trp 260 786 basepairs nucleic acid double linear Genomic DNA not provided CodingSequence 1...786 9 ATG AAA GAT GAT TTT GCA AAA CTT GAG GAA CAA TTT GATGCA AAA CTC 48 Met Lys Asp Asp Phe Ala Lys Leu Glu Glu Gln Phe Asp AlaLys Leu 1 5 10 15 GGG ATC TTT GCA TTG GAT ACA GGT ACA AAC CGG ACG GTAGCG TAT CGG 96 Gly Ile Phe Ala Leu Asp Thr Gly Thr Asn Arg Thr Val AlaTyr Arg 20 25 30 CCG GAT GAG CGT TTT GCT TTT GCT TCG ACG ATT AAG GCT TTAACT GTA 144 Pro Asp Glu Arg Phe Ala Phe Ala Ser Thr Ile Lys Ala Leu ThrVal 35 40 45 GGC GTG CTT TTG CAA CAG AAA TCA ATA GAA GAT CTG AAC CAG AGAATA 192 Gly Val Leu Leu Gln Gln Lys Ser Ile Glu Asp Leu Asn Gln Arg Ile50 55 60 ACA TAT ACA CGT GAT GAT CTT GTA AAC TAC AAC CCG ATT ACG GAA AAG240 Thr Tyr Thr Arg Asp Asp Leu Val Asn Tyr Asn Pro Ile Thr Glu Lys 6570 75 80 CAC GTT GAT ACG GGA ATG ACG CTC AAA GAG CTT GCG GAT GCT TCG CTT288 His Val Asp Thr Gly Met Thr Leu Lys Glu Leu Ala Asp Ala Ser Leu 8590 95 CGA TAT AGT GAC AAT GCG GCA CAG AAT CTC ATT CTT AAA CAA ATT GGC336 Arg Tyr Ser Asp Asn Ala Ala Gln Asn Leu Ile Leu Lys Gln Ile Gly 100105 110 GGA CCT GAA AGT TTG AAA AAG GAA CTG AGG AAG ATT GGT GAT GAG GTT384 Gly Pro Glu Ser Leu Lys Lys Glu Leu Arg Lys Ile Gly Asp Glu Val 115120 125 ACA AAT CCC GAA CGA TTC GAA CCA GAG TTA AAT GAA GTG AAT CCG GGT432 Thr Asn Pro Glu Arg Phe Glu Pro Glu Leu Asn Glu Val Asn Pro Gly 130135 140 GAA ACT CAG GAT ACC AGT ACA GCA AGA GCA CTT GTC ACA AGC CTT CGA480 Glu Thr Gln Asp Thr Ser Thr Ala Arg Ala Leu Val Thr Ser Leu Arg 145150 155 160 GCC TTT GCT CTT GAA GAT AAA CTT CCA AGT GAA AAA CGC GAG CTTTTA 528 Ala Phe Ala Leu Glu Asp Lys Leu Pro Ser Glu Lys Arg Glu Leu Leu165 170 175 ATC GAT TGG ATG AAA CGA AAT ACC ACT GGA GAC GCC TTA ATC CGTGCC 576 Ile Asp Trp Met Lys Arg Asn Thr Thr Gly Asp Ala Leu Ile Arg Ala180 185 190 GGA GCG GCA TCA TAT GGA ACC CGG AAT GAC ATT GCC ATC ATT TGGCCG 624 Gly Ala Ala Ser Tyr Gly Thr Arg Asn Asp Ile Ala Ile Ile Trp Pro195 200 205 CCA AAA GGA GAT CCT GTC GGT GTG CCG GAC GGT TGG GAA GTG GCTGAT 672 Pro Lys Gly Asp Pro Val Gly Val Pro Asp Gly Trp Glu Val Ala Asp210 215 220 AAA ACT GTT CTT GCA GTA TTA TCC AGC AGG GAT AAA AAG GAC GCCAAG 720 Lys Thr Val Leu Ala Val Leu Ser Ser Arg Asp Lys Lys Asp Ala Lys225 230 235 240 TAT GAT GAT AAA CTT ATT GCA GAG GCA ACA AAG GTG GTA ATGAAA GCC 768 Tyr Asp Asp Lys Leu Ile Ala Glu Ala Thr Lys Val Val Met LysAla 245 250 255 TTA AAC ATG AAC GGC AAA 786 Leu Asn Met Asn Gly Lys 260262 amino acids amino acid linear protein internal not provided 10 MetLys Asp Asp Phe Ala Lys Leu Glu Glu Gln Phe Asp Ala Lys Leu 1 5 10 15Gly Ile Phe Ala Leu Asp Thr Gly Thr Asn Arg Thr Val Ala Tyr Arg 20 25 30Pro Asp Glu Arg Phe Ala Phe Ala Ser Thr Ile Lys Ala Leu Thr Val 35 40 45Gly Val Leu Leu Gln Gln Lys Ser Ile Glu Asp Leu Asn Gln Arg Ile 50 55 60Thr Tyr Thr Arg Asp Asp Leu Val Asn Tyr Asn Pro Ile Thr Glu Lys 65 70 7580 His Val Asp Thr Gly Met Thr Leu Lys Glu Leu Ala Asp Ala Ser Leu 85 9095 Arg Tyr Ser Asp Asn Ala Ala Gln Asn Leu Ile Leu Lys Gln Ile Gly 100105 110 Gly Pro Glu Ser Leu Lys Lys Glu Leu Arg Lys Ile Gly Asp Glu Val115 120 125 Thr Asn Pro Glu Arg Phe Glu Pro Glu Leu Asn Glu Val Asn ProGly 130 135 140 Glu Thr Gln Asp Thr Ser Thr Ala Arg Ala Leu Val Thr SerLeu Arg 145 150 155 160 Ala Phe Ala Leu Glu Asp Lys Leu Pro Ser Glu LysArg Glu Leu Leu 165 170 175 Ile Asp Trp Met Lys Arg Asn Thr Thr Gly AspAla Leu Ile Arg Ala 180 185 190 Gly Ala Ala Ser Tyr Gly Thr Arg Asn AspIle Ala Ile Ile Trp Pro 195 200 205 Pro Lys Gly Asp Pro Val Gly Val ProAsp Gly Trp Glu Val Ala Asp 210 215 220 Lys Thr Val Leu Ala Val Leu SerSer Arg Asp Lys Lys Asp Ala Lys 225 230 235 240 Tyr Asp Asp Lys Leu IleAla Glu Ala Thr Lys Val Val Met Lys Ala 245 250 255 Leu Asn Met Asn GlyLys 260

What is claimed is:
 1. A method of determining the amount ofbeta-lactamase activity in a cell, comprising the steps of: a) providinga mammalian host cell transfected with a recombinant nucleic acidmolecule comprising an expression control sequence adapted for functionin a mammalian cell operatively linked to a nucleic acid sequence codingfor the expression of a cytosolic form of beta-lactamase, wherein saidnucleotide sequence encoding a cytosolic form of beta-lactamase lacks anucleotide sequence encoding an amino acid sequence for secretion, b)contacting a sample comprising said mammalian host cell or an extract ofsaid mammalian host cell with a substrate for beta-lactamase, and c)determining the amount of said substrate for beta-lactamase cleaved,whereby the amount of said substrate for beta-lactamase cleaved isrelated to the amount of beta-lactamase activity.
 2. A method formonitoring the expression of a mammalian gene, comprising: a) providinga mammalian host cell transfected with a recombinant nucleic acidmolecule comprising an expression control sequence adapted for functionin said mammalian host cell operatively linked to a nucleic acidsequence coding for the expression of a cytosolic form ofbeta-lactamase, wherein said nucleotide sequence encoding a cytosolicform of beta-lactamase lacks a nucleotide sequence encoding an aminoacid sequence for secretion, b) contacting a sample comprising saidmammalian host cell or an extract of said mammalian host cell with asubstrate for beta-lactamase, and c) determining cleavage of thesubstrate, whereby the cleavage of substrate indicates expression.
 3. Amethod for determining whether a test compound alters the expression ofa gene operably linked to a set of expression control sequences,comprising: a) providing a cell transfected with a recombinant nucleicacid construct comprising an expression control sequence adapted forfunction in said cell operably linked to a nucleic acid sequence codingfor the expression of a cytosolic form of beta-lactamase, wherein saidnucleotide sequence encoding a cytosolic form of beta-lactamase lacks anucleotide sequence encoding an amino acid sequence for secretion, b)contacting the cell with the test compound, c) contacting a samplecomprising said cell or an extract of said cell with a beta-lactamasesubstrate, and d) determining cleavage of said beta-lactamase substrate,whereby the cleavage of said beta-lactamase substrate indicates whetherthe test compound alters expression.
 4. A recombinant nucleic acidmolecule, comprising: an expression control sequence adapted forfunction in a vertebrate cell operably linked to a nucleotide sequenceencoding a cytosolic form of beta-lactamase, wherein said nucleotidesequence encoding a cytosolic form of beta-lactamase lacks a nucleotidesequence encoding an amino acid sequence for secretion.
 5. A recombinantnucleic acid molecule, comprising: an expression control sequenceadapted for function in an eukaryotic cell operably linked to apolynucleotide comprising a nucleotide sequence encoding a cytosolicbeta-lactamase, wherein said nucleotide sequence encoding a cytosolicform of beta-lactamase lacks a nucleotide sequence encoding an aminoacid sequence for secretion.
 6. A cell, comprising a mammalian host celland a recombinant nucleic acid molecule, comprising: an expressioncontrol sequence adapted for function in a mammalian cell and operablylinked to a polynucleotide comprising a nucleotide sequence encoding acytosolic form of beta-lactamase, wherein said nucleotide sequenceencoding a cytosolic form of beta-lactamase lacks a nucleotide sequenceencoding an amino acid sequence for secretion.
 7. A recombinant nucleicacid molecule, comprising: a nucleic acid molecule encoding a cytosolicbeta-lactamase operatively linked to a mammalian expression controlsequence, wherein said nucleotide sequence encoding a cytosolic form ofbeta-lactamase lacks a nucleotide sequence encoding an amino acidsequence for secretion.
 8. The recombinant nucleic acid molecule ofclaim 7, wherein said recombinant nucleic acid molecule can becontrollably expressed in a mammalian cell.
 9. The recombinant nucleicacid molecule of claim 8, wherein said nucleic acid molecule encoding acytosolic beta-lactamase encodes a cytosolic beta-lactamase that is notpart of a fusion protein.
 10. The recombinant nucleic acid molecule ofclaim 7, wherein said mammalian expression control sequence isresponsive to substances that modulate intra-cellular receptors.
 11. Therecombinant nucleic acid molecule of claim 7, wherein said mammalianexpression control sequence is responsive to substances that modulatecell-surface receptors.
 12. A mammalian cell, comprising: a recombinantnucleic acid molecule encoding a cytosolic form of a beta-lactamaseoperatively linked to a mammalian expression control sequence, whereinsaid beta-lactamase is functional and localized in the cytosol of saidmammalian cell.
 13. The mammalian cell of claim 12, wherein saidrecombinant nucleic acid molecule is introduced into the genome of saidmammalian cell.
 14. The mammalian cell of claim 13, wherein saidrecombinant nucleic acid molecule does not encode a fusion protein. 15.The mammalian cell of claim 12, wherein said mammalian cell furthercomprises a cell-membrane-permeable beta-lactamase substrate or esterderivative thereof.
 16. The mammalian cell of claim 15, wherein saidbeta-lactamase substrate or ester derivative thereof is transformed bysaid mammalian cell in to a compound that is lesscell-membrane-permeable than said beta-lactamase substrate or esterderivative thereof.
 17. The mammalian cell of claim 16, wherein saidcell-membrane-permeable beta-lactamase substrate comprises a fluorescentdonor and a fluorescent acceptor.
 18. The mammalian cell of claim 12,wherein said mammalian expression control sequence is responsive tosubstances that modulate intra-cellular receptors.
 19. The mammaliancell of claim 12, wherein said mammalian expression control sequence isresponsive to substances that modulate cell-surface receptors.
 20. Themammalian cell of claim 12, wherein said cell further comprises a testcompound.
 21. A recombinant nucleic acid molecule, comprising anucleotide sequence encoding a non-secreted form of beta-lactamase,wherein said nucleotide sequence encoding a non-secreted form ofbeta-lactamase is operatively linked to a mammalian expression controlsequence that is responsive to substances that modulate cell surfacereceptors.
 22. A mammalian expression vector, comprising a recombinantnucleic acid molecule, comprising a nucleotide sequence encoding anon-secreted form of beta lactamase wherein said nucleotide sequenceencoding a non-secreted form of beta lactamase is operatively linked toa mammalian expression control sequence.
 23. The mammalian expressionvector of claim 22, wherein said mammalian expression control sequenceis responsive to substances that modulate cell surface receptors. 24.The vector of claim 22, further comprising a Kozak sequence.